Compositions and methods for treating cancer and diseases and conditions responsive to cell growth inhibition

ABSTRACT

In alternative embodiments, the invention provides compositions and methods for overcoming or diminishing or preventing Growth Factor Inhibitor resistance in a cell, or, a method for increasing the growth-inhibiting effectiveness of a Growth Factor inhibitor on a cell, or, a method for re-sensitizing a cell to a Growth Factor Inhibitor, comprising for example, administration of a combination of a TBK1 inhibitor and an RTK inhibitor. In alternative embodiments, the cell is a tumor cell, a cancer cell or a dysfunctional cell. In alternative embodiments, the invention provides compositions and methods for determining: whether an individual or a patient would benefit from or respond to administration of a Growth Factor Inhibitor, or, which individuals or patients would benefit from a combinatorial approach comprising administration of a combination of: at least one growth factor and at least one compound, composition or formulation used to practice a method of the invention, such as an NFKB inhibitor, such as a lenalidomide or a REVLIMID™, or IKK inhibitor; or an inhibitor of Galectin-3.

RELATED APPLICATIONS

This application is a continuation in part (CIP) of Patent ConventionTreaty (PCT) International Application Serial No: PCT/US2013/035492,filed Apr. 5, 2013, now pending, which claims benefit of priority toInternational Application Serial No: PCT/US2012/040390, filed Jun. 2,2012, and which also claims benefit of priority to U.S. ProvisionalPatent Application Ser. No. 61/672,236, filed Jul. 16, 2012, and U.S.Ser. No. 61/620,725, filed Apr. 5, 2012. This application also claimsthe benefit of priority under 35 U.S.C. §119(e) of U.S. ProvisionalPatent Application Ser. No. 61/843,417, filed Jul. 7, 2013. Theaforementioned applications are expressly incorporated herein byreference in their entirety and for all purposes.

GOVERNMENT RIGHTS

This invention was made with government support under grant numbersCA045726, CA050286, CA095262, HL057900, and HL103956, awarded by theNational Institutes of Health (NIH). The government has certain rightsin the invention.

TECHNICAL FIELD

This invention generally relates to cell and molecular biology,diagnostics and oncology. In alternative embodiments, the inventionprovides compositions and methods for overcoming or diminishing orpreventing Growth Factor Inhibitor resistance in a cell, or, a methodfor increasing the growth-inhibiting effectiveness of a Growth Factorinhibitor on a cell, or, a method for re-sensitizing a cell to a GrowthFactor Inhibitor, or, sensitizing a tumor to a drug, wherein optionallythe drug is an erlotinib or a lapatinib, or, sensitizing a tumor that isresistant to a cancer drug, comprising for example, administration of acombination of a TBK1 inhibitor and an RTK inhibitor. In alternativeembodiments, the cell is a tumor cell, a cancer cell, a cancer stem cellor a dysfunctional cell. In alternative embodiments, the inventionprovides compositions and methods for determining: whether an individualor a patient would benefit from or respond to administration of a GrowthFactor Inhibitor, or, which individuals or patients would benefit from acombinatorial approach comprising administration of a combination of: atleast one growth factor and at least one compound, composition orformulation used to practice a method of the invention, such as an NFKB(nuclear factor kappa-light-chain-enhancer of activated B cells, orNF-κB) inhibitor, such as a lenalidomide or a REVLIMID™, or an IκBkinase (IKK) inhibitor; or an inhibitor of Galectin-3.

BACKGROUND

Growth factor inhibitors have been used to treat many cancers includingpancreatic, breast, lung and colorectal cancers. However, resistance togrowth factor inhibitors has emerged as a significant clinical problem.

Tumor resistance to targeted therapies occurs due to a combination ofstochastic and instructional mechanisms. Mutation/amplification intyrosine kinase receptors or their downstream effectors account for theresistance of a broad range of tumors. In particular, oncogenic KRAS,the most commonly mutated oncogene in human cancer, has been linked toEGFR inhibitor resistance. However, in lung and pancreatic carcinomas,recent studies suggest that oncogenic KRAS is not sufficient to accountfor EGFR inhibitor resistance indicating that other factor(s) mightcontrol this process.

SUMMARY

In alternative embodiments, the invention provides methods for:

overcoming or diminishing or preventing a Growth Factor Inhibitor (GFI)resistance in a cell, or

increasing the growth-inhibiting effectiveness of a Growth Factorinhibitor on a cell, or

sensitizing, increasing sensitivity to or re-sensitizing a cell to aGrowth Factor Inhibitor (GFI), or

sensitizing, increasing sensitivity to or re-sensitizing a dysfunctionalcell, a tumor or cancer to a drug,

-   -   wherein optionally the drug is a Receptor Tyrosine Kinase (RTK)        inhibitor, an EGFR1 inhibitor, an EGFR1/EGFR2 inhibitor or an        IGF-1R inhibitor, or an erlotinib, a linsitinib, a lapatinib or        a lenalidomide,

sensitizing, increasing sensitivity to or re-sensitizing a tumor that isresistant to a cancer or anti-tumor drug, or

reversing a tumor cell, a cancer cell, a cancer stem cell or adysfunctional cell initiation or self-renewal capacity,

-   -   wherein optionally the cell is a tumor cell, a cancer cell, a        cancer stem cell, or a dysfunctional cell,

the method comprising:

(a)(1) providing at least one compound, composition or formulationcomprising or consisting of:

-   -   (i) an inhibitor or depleter of integrin α_(v)β₃ (anb3), or    -   an inhibitor of integrin α_(v)β₃ (anb3) protein activity, or    -   an inhibitor of the formation or activity of an integrin        anb3/RalB signaling complex, or    -   an inhibitor of the formation or signaling activity of an        integrin α_(v)β₃ (anb3)/RalB/NFkB signaling axis,    -   wherein optionally the inhibitor of integrin α_(v)β₃ protein        activity is an allosteric inhibitor of integrin α_(v)β₃ protein        activity;    -   (ii) an inhibitor or depleter of a RalB protein or an inhibitor        of a RalB protein activation, or    -   an inhibitor or depleter of the recruitment of KRAS/RalB to the        plasma membrane or the association of KRAS to RalB,    -   wherein optionally the inhibitor is an allosteric inhibitor, or    -   optionally the inhibitor of the RalB protein activity is an        allosteric inhibitor of RalB protein activity;    -   (iii) an inhibitor or depleter of a Src or a Tank Binding        Kinase-1 (TBK1) protein or an inhibitor of Src or TBK1 protein        activation,    -   wherein optionally the inhibitor of the Src or the TBK1 protein        activity is:    -   an amlexanox (or        2-amino-7-isopropyl-5-oxo-5H-chromeno[2,3-b]pyridine-3-carboxylic        acid), or an APHTHASOL™; or    -   a γ(1)34.5 protein of herpes simplex viruses (HSV) (see e.g., Ma        et al., J Virol. 2012 February; 86(4):2188-96); or    -   a BX795 as described in, e.g., Bain et al., Biochem J. (2007)        Dec. 15; 408(3):297-315; Clark et al., (2009) J. Biol. Chem.        284:14136-14146; or    -   an azabenzimidazole or an analog or derivative thereof; or    -   a 6-amino-pyrazolopyrimidine or an analog or derivative thereof;        or,    -   a compound having one of the following formulas, or an analog or        derivative thereof (see Hutti, et al., (2012) Development of a        High-Throughput Assay for Identifying Inhibitors of TBK1 and        IKKε. PLoS ONE 7(7):e4149doi: 10.1371/journal.pone):

Molecule IKKα TBK1 IKKβ IKKα

0.77 0.44 >10 >10

>10 0.50 >10 >10

>10 0.64 8.76 >10

>10 0.67 >10 >10

>10 0.87 >10 >10

-   -   and optionally the inhibitor of the Src or the TBK1 protein        activity is an allosteric inhibitor of Src or TBK1 protein        activity;    -   (iv) an inhibitor or depleter of a NFKB or a Interferon        regulatory factor 3 (IRF3) protein or an inhibitor of RalB        protein activation,    -   wherein optionally the inhibitor of the NFKB or the IRF3 protein        activity is an allosteric inhibitor of an NFKB or an Interferon        regulatory factor 3 (IRF3) protein activity;    -   (v) an inhibitor or depleter of NFKB or IKK, or an inhibitor of        NFKB or IKK protein activation,    -   wherein optionally the NFKB inhibitor comprises a lenalidomide        or a REVLIMID™ (Celgene Corp., Summit, N.J.) and optionally the        IKK inhibitor comprises a PS1145 (Millennium Pharmaceuticals,        Cambridge, Mass.);    -   (vi) a lenalidomide or a REVLIMID™ and PS1145;    -   (vii) a lenalidomide or a REVLIMID™; a PS1145; and, a Receptor        Tyrosine Kinase (RTK) inhibitor, and optionally the RTK        inhibitor comprises SU14813 (Pfizer, San Diego, Calif.);    -   (viii) an inhibitor of Galectin-3; or    -   (ix) any combination of (i) to (viii), or

(2) one or any combination of the compound, composition or formulation,or compounds, compositions or formulations, of (1), and at least onegrowth factor inhibitor,

wherein optionally the at least one growth factor inhibitor comprises aReceptor Tyrosine Kinase (RTK) inhibitor, a Src inhibitor, ananti-metabolite inhibitor, a gemcitabine, a GEMZAR™, a mitotic poison, apaclitaxel, a taxol, an ABRAXANE™, an erlotinib, a TARCEVA™, alapatinib, a TYKERB™, a cetuxamib, an ERBITUX™, or an insulin growthfactor inhibitor;

wherein optionally the combination or the therapeutic combinationcomprises: (i) an inhibitor or depleter of a Src or a Tank BindingKinase-1 (TBK1) protein or an inhibitor of Src or TBK1 proteinactivation, wherein optionally the inhibitor of the Src or the TBK1protein activity is an amlexanox (or2-amino-7-isopropyl-5-oxo-5H-chromeno[2,3-b]pyridine-3-carboxylic acid)or APHTHASOL™, and (ii) an RTK inhibitor, wherein optionally the RTKinhibitor is a Src inhibitor, an anti-metabolite inhibitor, agemcitabine, a GEMZAR™, a mitotic poison, a paclitaxel, a taxol, anABRAXANE™, an erlotinib, a TARCEVA™, a lapatinib, a TYKERB™, acetuxamib, an ERBITUX™, or an insulin growth factor inhibitor or acombination thereof;

wherein optionally the combination or the therapeutic combinationcomprises an erlotinib with either a Lenalidomide or a PS-1145, or botha Lenalidomide and a PS-1145; and

(b) administering a sufficient amount of the at least one compound,composition or formulation to the cell, or the combination of compounds,to:

overcome or diminish or prevent a Growth Factor Inhibitor (GFI)resistance in a cell, or

increase the growth-inhibiting effectiveness of a Growth Factorinhibitor on a cell, or

sensitize, increase sensitivity or re-sensitize a cell to a GrowthFactor Inhibitor (GFI), or

sensitize, increase sensitivity or re-sensitize a dysfunctional cell, atumor or cancer to a drug, wherein optionally the drug is a ReceptorTyrosine Kinase (RTK) inhibitor, or an erlotinib, a lapatinib or alenalidomide,

sensitize, increase sensitivity or re-sensitize a tumor that isresistant to a cancer or anti-tumor drug, or

reverse a tumor cell, a cancer cell, a cancer stem cell or adysfunctional cell initiation or self-renewal capacity.

In alternative embodiments of the methods:

(a) the at least one compound, composition or formulation, orcombination of compounds, is formulated as a pharmaceutical composition;

(b) the method of (a), wherein the compound, composition or formulationor pharmaceutical composition is administered in vitro, ex vivo or invivo, or is administered to an individual in need thereof;

(c) the method of (a) or (b), wherein the at least one compound,composition or formulation is a pharmaceutical composition is formulatedfor administration intravenously (IV), parenterally, nasally, topically,orally, or by liposome or targeted or vessel-targeted nanoparticledelivery;

(d) the method of any of (a) to (c), wherein the compound or compositioncomprises or is an inhibitor of transcription, translation or proteinexpression;

(e) the method of any of (a) to (d), wherein the compound or compositionis a small molecule, a protein, an antibody, a monoclonal antibody, anucleic acid, a lipid or a fat, a polysaccharide, an RNA or a DNA;

(f) the method of any of (a) to (e), wherein the compound or compositioncomprises or is: a VITAXIN™ (Applied Molecular Evolution, San Diego,Calif.) antibody, a humanized version of an LM609 monoclonal antibody,an LM609 monoclonal antibody, or any antibody that functionally blocksan α_(v)β₃ integrin or any member of an α_(v)β₃ integrin-comprisingcomplex or an integrin α_(v)β₃ (anb3)/RalB/NFkB signaling axis;

(g) the method of any of (a) to (e), wherein the compound or compositioncomprises or is a Src inhibitor, a dasatinib, a saracatinib; abosutinib; a NVP-BHG712, or any combination thereof;

(h) the method of any of (a) to (g), wherein Growth Factor Inhibitor isor comprises an anti-metabolite inhibitor, a gemcitabine, GEMZAR™, amitotic poison, a paclitaxel, a taxol, ABRAXANE™, an erlotinib,TARCEVA™, a lapatinib, TYKERB™, or an insulin growth factor inhibitor,or any combination thereof;

(i) the method of any of (a) to (h), wherein the Growth Factor Inhibitordecreases, slows or blocks new blood vessel growth, neovascularizationor angiogenesis; or, wherein administering the Growth Factor Inhibitortreats or ameliorates conditions that are responsive to blocking orslowing cell growth, and/or the development of neovascularization or newblood vessels;

(j) the method of any of (a) to (h), wherein the NF-kB inhibitorcomprises or consists of one or more of: an antioxidant; an α-lipoicacid; an α-tocopherol; a2-amino-1-methyl-6-phenylimidazo[4,5-β]pyridine; an allopurinol; ananetholdithiolthione; a cepharanthine; a beta-carotene; adehydroepiandrosterone (DHEA) or a DHEA-sulfate (DHEAS); adimethyldithiocarbamates (DMDTC); a dimethylsulfoxide (DMSO); a flavone,a Glutathione; Vitamin C or Vitamin B6, or one or more compositionslisted in Table 1 or Table 2, or any combination thereof;

-   -   (k) the method of any of (a) to (j), wherein the at least one        compound, composition or formulation, or combination of        compounds, comprises a protcasome inhibitor or a protease        inhibitor that can inhibit an Rei and/or an NFkB, or one or more        compositions listed in Table 2, or any combination thereof;    -   (l) the method of any of (a) to (j), wherein the at least one        compound, composition or formulation, or combination of        compounds, comprises an IκBα (nuclear factor of kappa light        polypeptide gene enhancer in B-cells inhibitor, alpha)        phosphorylation and/or degradation inhibitor, or one or more        compositions listed in Table 3, or any combination thereof; or    -   (m) the method of any of (a) to (l), wherein the method reduces,        treats or ameliorates the level of disease in a retinal        age-related macular degeneration, a diabetic retinopathy, a        cancer or carcinoma, a glioblastoma, a neuroma, a neuroblastoma,        a colon carcinoma, a hemangioma, an infection and/or a condition        with at least one inflammatory component, and/or any infectious        or inflammatory disease, such as a rheumatoid arthritis, a        psoriasis, a fibrosis, leprosy, multiple sclerosis, inflammatory        bowel disease, or ulcerative colitis or Crohn's disease.

In alternative embodiments, the invention provides kits, blisterpackages, lidded blisters or blister cards or packets, clamshells, traysor shrink wraps, comprising;

(a)(i) at least one compound, composition or formulation used topractice a method of the invention, and (ii); at least one Growth FactorInhibitor; or

(b) the kit of (a), further comprising instructions for practicing amethod of the invention.

In alternative embodiments, the kit, blister package, lidded blister,blister card, packet, clamshell, tray or shrink wrap comprises: acombination or a therapeutic combination of drugs comprising: anerlotinib with either a Lenalidomide or a PS-1145, or both aLenalidomide and a PS-1145.

In alternative embodiments, the invention provides methods fordetermining:

whether an individual or a patient would benefit from or respond toadministration of a Growth Factor Inhibitor, or

which individuals or patients would benefit from a combinatorialapproach comprising administration of a combination of: at least onegrowth factor and at least one compound, composition or formulation usedto practice a method of the invention, such as an NfKb inhibitor,

the method comprising:

detecting the levels or amount of integrin α_(v)β₃ (anb3) and/or activeRalB complex in or on a cell, a tissue or a tissue sample,

wherein optionally the detection is by analysis or visualization of abiopsy or a tissue, urine, fluid, serum or blood sample, or a pathologyslide taken from the patient or individual, or by afluorescence-activated cell sorting (FACS) or flow cytometry analysis orthe sample or biopsy,

wherein optionally the cell or tissue or tissue sample is or is derivedfrom a tumor or a cancer,

wherein optionally the method further comprises taking a biopsy or atissue, urine, fluid, serum or blood sample from an individual or apatient,

wherein a finding of increased levels or amounts of integrin α_(v)β₃(anb3) and/or active RalB complexes in or on the cell, tissue or thetissue sample as compared to normal, normalized or wild type cells ortissues, indicates that:

the individual or patient would benefit from a combinatorial approachcomprising administration of a combination of: at least one growthfactor and at least one compound, composition or formulation used topractice a method of the invention.

In alternative embodiments of methods of the invention, the detecting ofthe levels or amount of integrin α_(v)β₃ (anb3) and/or active RalBcomplex in or on the cell, tissue or the tissue sample is done before orduring a drug or a pharmaceutical treatment of an individual using atleast one compound, composition or formulation used to practice a methodof the invention.

In alternative embodiments, the invention provide uses of a combinationof compounds in the manufacture of a medicament,

wherein the combination of compounds comprises:

(1) at least one compound comprising or consisting of:

-   -   (i) an inhibitor or depleter of integrin α_(v)β₃ (anb3), or an        inhibitor of integrin α_(v)β₃ (anb3) protein activity, or an        inhibitor of the formation or activity of an integrin anb3/RalB        signaling complex, or    -   an inhibitor of the formation or signaling activity of an        integrin α_(v)β₃ (anb3)/RalB/NFkB signaling axis,    -   wherein optionally the inhibitor of integrin α_(v)β₃ protein        activity is an allosteric inhibitor of integrin α_(v)β₃ protein        activity;    -   (ii) an inhibitor or depleter of a RalB protein or an inhibitor        of a RalB protein activation, or an inhibitor or depleter of the        recruitment of KRAS/RalB to the plasma membrane or the        association of KRAS to RalB,    -   wherein optionally the inhibitor is an allosteric inhibitor, or        the inhibitor of the RalB protein activity is an allosteric        inhibitor of RalB protein activity;    -   (iii) an inhibitor or depleter of a Src or a Tank Binding Kinase        (TBK1) protein or an inhibitor of Src or TBK1 protein        activation,    -   wherein optionally the inhibitor of the Src or the TBK1 protein        activity is: an amlexanox (or        2-amino-7-isopropyl-5-oxo-5H-chromeno[2,3-b]pyridine-3-carboxylic        acid), or an APHTHASOL™; or    -   a γ(1)34.5 protein of herpes simplex viruses (HSV) (see e.g., Ma        et al., J Virol. 2012 February; 86(4):2188-96); or, BX795 (as        described in, e.g., Bain et al., Biochem J. (2007) Dec. 15;        408(3):297-315; Clark et al., (2009) J. Biol. Chem.        284:14136-14146); or    -   an azabenzimidazole or an analog or derivative thereof; or a        6-amino-pyrazolopyrimidine or an analog or derivative thereof;        or,    -   a compound having one of the following formulas, or an analog or        derivative thereof (see Hutti, et al., (2012) Development of a        High-Throughput Assay for Identifying inhibitors of TBK1 and        IKKε. PLoS ONE 7(7):e411494.doi: 10.1371/joural.pone):

Molecule IKKε TBK1 IKKβ IKKα

0.77 0.44 >10 >10

>10 0.50 >10 >10

>10 0.64 8.76 >10

>10 0.67 >10 >10

>10 0.87 >10 >10

-   -   and optionally the inhibitor of the Src or the TBK1 protein        activity is an allosteric inhibitor of Src or TBK1 protein        activity;    -   (iv) an inhibitor or depleter of a NFKB or a Interferon        regulatory factor 3 (IRF3) protein or an inhibitor of RalB        protein activation,    -   wherein optionally the inhibitor of the NFKB or the IRF3 protein        activity is an allosteric inhibitor of an NFKB or an Interferon        regulatory factor 3 (IRF3) protein activity;    -   (v) an inhibitor or depleter of NFKB or IKK, or an inhibitor of        NFKB or IKK protein activation,    -   wherein optionally the NFKB inhibitor comprises a lenalidomide        or a REVLIMID™ (Celgene Corp., Summit, N.J.) and optionally the        IKK inhibitor comprises a PS1145 (Millennium Pharmaceuticals,        Cambridge, Mass.);    -   (vi) a lenalidomide or a REVLIMID™ and PS1145;    -   (vii) a lenalidomide or a REVLIMID™; a PS1145; and, a Receptor        Tyrosine Kinase (RTK) inhibitor, and optionally the RTK        inhibitor comprises SU14813 (Pfizer, San Diego, Calif.);    -   (viii) an inhibitor of Galectin-3; or    -   (ix) any combination of (i) to (viii), or

(2) one or any combination of the compound, composition or formulation,or compounds, compositions or formulations, of (1), and at least onegrowth factor inhibitor,

wherein optionally the at least one growth factor inhibitor comprises aReceptor Tyrosine Kinase (RTK) inhibitor, a Src inhibitor, ananti-metabolite inhibitor, a gemcitabine, a GEMZAR™, a mitotic poison, apaclitaxel, a taxol, an ABRAXANE™, an erlotinib, a TARCEVA™, alapatinib, a TYKERB™, a cetuxamib, an ERBITUX™, or an insulin growthfactor inhibitor;

wherein optionally the combination or the therapeutic combinationcomprises: (i) an inhibitor or depleter of a Src or a Tank BindingKinase-1 (TBK1) protein or an inhibitor of Src or TBK1 proteinactivation, wherein optionally the inhibitor of the Src or the TBK1protein activity is an amlexanox (or2-amino-7-isopropyl-5-oxo-5H-chromeno[2,3-b]pyridine-3-carboxylic acid)or APHTHASOL™, and (ii) an RTK inhibitor, wherein optionally the RTKinhibitor is a Src inhibitor, an anti-metabolite inhibitor, agemcitabine, a GEMZAR™, a mitotic poison, a paclitaxel, a taxol, anABRAXANE™, an erlotinib, a TARCEVA™, a lapatinib, a TYKERB™, acetuxamib, an ERBITUX™, or an insulin growth factor inhibitor or acombination thereof;

wherein optionally the combination or the therapeutic combinationcomprises an erlotinib with either a Lenalidomide or a PS-1145, or botha Lenalidomide and a PS-1145;

(vii) a lenalidomide or a REVLIMID™; a PS1145; and, a Receptor TyrosineKinase (RTK) inhibitor, and optionally the RTK inhibitor comprisesSU14813 (Pfizer, San Diego, Calif.);

(viii) an inhibitor of Galectin-3; or

(ix) any combination of (i) to (viii); and

(2) at least one Growth Factor Inhibitor,

wherein optionally the Growth Factor Inhibitor is or comprises ananti-metabolite inhibitor, a gemcitabine, GEMZAR™, a mitotic poison, apaclitaxel, a taxol, ABRAXANE™, an erlotinib, TARCEVA™, a lapatinib,TYKERB™, or an insulin growth factor inhibitor, or any combinationthereof; or, the Growth Factor Inhibitor decreases, slows or blocks newblood vessel growth, neovascularization or angiogenesis; or, whereinadministering the Growth Factor Inhibitor treats or amelioratesconditions that are responsive to blocking or slowing cell growth,and/or the development of neovascularization or new blood vessels,

wherein optionally the combination or the therapeutic combinationcomprises an erlotinib with either a Lenalidomide or a PS-1145, or botha Lenalidomide and a PS-1145.

In alternative embodiments, the invention provides therapeuticcombinations of drugs comprising or consisting of a combination of atleast two compounds: wherein the at least two compounds comprise orconsist of:

(1) at least one compound comprising or consisting of:

-   -   (i) an inhibitor or depleter of integrin α_(v)β₃ (anb3), or an        inhibitor of integrin α_(v)β₃ (anb3) protein activity, or an        inhibitor of the formation or activity of an integrin anb3/RalB        signaling complex, or an inhibitor of the formation or signaling        activity of an integrin α_(v)β₃ (anb3)/RalB/NFkB signaling axis,    -   wherein optionally the inhibitor of integrin α_(v)β₃ protein        activity is an allosteric inhibitor of integrin α_(v)β₃ protein        activity;    -   (ii) an inhibitor or depleter of a RalB protein or an inhibitor        of a RalB protein activation, or an inhibitor or depleter of the        recruitment of KRAS/RalB to the plasma membrane or the        association of KRAS to RalB,    -   wherein optionally the inhibitor is an allosteric inhibitor, or        the inhibitor of the RalB protein activity is an allosteric        inhibitor of RalB protein activity;    -   (iii) an inhibitor or depleter of a Src or a Tank Binding Kinase        (TBK1) protein or an inhibitor of Src or TBK1 protein        activation,    -   wherein optionally the inhibitor of the Src or the TBK1 protein        activity is: an amlexanox (or        2-amino-7-isopropyl-5-oxo-5H-chromeno[2,3-b]pyridine-3-carboxylic        acid), or an APHTHASOL™; or a γ(1)34.5 protein of herpes simplex        viruses (HSV) (see e.g., Ma et al., J Virol. 2012 February;        86(4):2188-96); or, BX795 (as described in, e.g., Bain et al.,        Biochem J. (2007) Dec. 15; 408(3):297-315; Clark et        al., (2009) J. Biol. Chem. 284:14136-14146); or an        azabenzimidazole or an analog or derivative thereof; or a        6-amino-pyrazolopyrimidine or an analog or derivative thereof;        or, a compound having one of the following formulas, or an        analog or derivative thereof (see Hatti, et al., (2012)        Development of a High-Throughput Assay for identifying        Inhibitors of TBK1 and IKKε. PLoS ONE 7(7):e41494.doi:        10.1371/journal.pone):

Molecule IKKε TBK1 IKKβ IKKα

0.77 0.44 >10 >10

>10 0.50 >10 >10

>10 0.64 8.76 >10

>10 0.67 >10 >10

>10 0.87 >10 >10

-   -   and optionally the inhibitor of the Src or the TBK1 protein        activity is an allosteric inhibitor of Src or TBK1 protein        activity;    -   (iv) an inhibitor or depleter of a NFKB or a Interferon        regulatory factor 3 (IRF3) protein or an inhibitor of RalB        protein activation,    -   wherein optionally the inhibitor of the NFKB or the IRF3 protein        activity is an allosteric inhibitor of an NFKB or an Interferon        regulatory factor 3 (IRF3) protein activity;    -   (v) an inhibitor or depleter of NFKB or IKK, or an inhibitor of        NFKB or IKK protein activation,    -   wherein optionally the NFKB inhibitor comprises a lenalidomide        or a REVLIMID™ (Celgene Corp., Summit, N.J.) and optionally the        IKK inhibitor comprises a PS1145 (Millennium Pharmaceuticals,        Cambridge, Mass.);    -   (vi) a lenalidomide or a REVLIMID™ and PS1145;    -   (vii) a lenalidomide or a REVLIMID™; a PS1145; and, a Receptor        Tyrosine Kinase (RTK) inhibitor, and optionally the RTK        inhibitor comprises SU14813 (Pfizer, San Diego, Calif.);    -   (viii) an inhibitor of Galectin-3; or    -   (ix) any combination of (i) to (viii), or

(2) one or any combination of the compound, composition or formulation,or compounds, compositions or formulations, of (1), and at least onegrowth factor inhibitor,

wherein optionally the at least one growth factor inhibitor comprises aReceptor Tyrosine Kinase (RTK) inhibitor, a Src inhibitor, ananti-metabolite inhibitor, a gemcitabine, a GEMZAR™, a mitotic poison, apaclitaxel, a taxol, an ABRAXANE™, an erlotinib, a TARCEVA™, alapatinib, a TYKERB™, a cetuxamib, an ERBITUX™, or an insulin growthfactor inhibitor;

wherein optionally the combination or the therapeutic combinationcomprises: (i) an inhibitor or depleter of a Src or a Tank BindingKinase-1 (TBK1) protein or an inhibitor of Src or TBK1 proteinactivation, wherein optionally the inhibitor of the Src or the TBK1protein activity is an amlexanox (or2-amino-7-isopropyl-5-oxo-5H-chromeno[2,3-b]pyridine-3-carboxylic acid)or APHTHASOL™, and (ii) an RTK inhibitor, wherein optionally the RTKinhibitor is a Src inhibitor, an anti-metabolite inhibitor, agemcitabine, a GEMZAR™, a mitotic poison, a paclitaxel, a taxol, anABRAXANE™, an erlotinib, a TARCEVA™, a lapatinib, a TYKERB™, acetuxamib, an ERBITUX™, or an insulin growth factor inhibitor or acombination thereof;

wherein optionally the combination or the therapeutic combinationcomprises an erlotinib with either a Lenalidomide or a PS-1145, or botha Lenalidomide and a PS-1145;

(vii) a lenalidomide or a REVLIMID™; a PS1145; and, a Receptor TyrosineKinase (RTK) inhibitor, and optionally the RTK inhibitor comprisesSU14813 (Pfizer, San Diego, Calif.);

(viii) an inhibitor of Galectin-3; or

(ix) any combination of (i) to (viii); and

(2) at least one Growth Factor Inhibitor,

wherein optionally the Growth Factor Inhibitor is or comprises ananti-metabolite inhibitor, a gemcitabine, GEMZAR™, a mitotic poison, apaclitaxel, a taxol, ABRAXANE™, an erlotinib, TARCEVA™, a lapatinib,TYKERB™, or an insulin growth factor inhibitor, or any combinationthereof; or, the Growth Factor Inhibitor decreases, slows or blocks newblood vessel growth, neovascularization or angiogenesis; or,

wherein administering the Growth Factor Inhibitor treats or amelioratesconditions that are responsive to blocking or slowing cell growth,and/or the development of neovascularization or new blood vessels,

wherein optionally the combination or the therapeutic combinationcomprises an erlotinib with either a Lenalidomide or a PS-1145, or botha Lenalidomide and a PS-1145.

In alternative embodiments, the invention provides combinations, ortherapeutic combinations, for overcoming or diminishing or preventingGrowth Factor Inhibitor (GFI) resistance in a cell, or, a method forincreasing the growth-inhibiting effectiveness of a Growth Factorinhibitor on a cell, or, a method for re-sensitizing a cell to a GrowthFactor Inhibitor (GFI), wherein the combination comprises or consistsof:

(1) at least one compound comprising or consisting of:

-   -   (i) an inhibitor or depleter of integrin α_(v)β₃ (anb3), or an        inhibitor of integrin α_(v)β₃ (anb3) protein activity, or an        inhibitor of the formation or activity of an integrin anb3/RalB        signaling complex, or an inhibitor of the formation or signaling        activity of an integrin α_(v)β₃ (anb3)/RalB/NFkB signaling axis,    -   wherein optionally the inhibitor of integrin α_(v)β₃ protein        activity is an allosteric inhibitor of integrin α_(v)β₃ protein        activity;    -   (ii) an inhibitor or depleter of a RalB protein or an inhibitor        of a RalB protein activation, or an inhibitor or depleter of the        recruitment of KRAS/RalB to the plasma membrane or the        association of KRAS to RalB,    -   wherein optionally the inhibitor is an allosteric inhibitor, or        the inhibitor of the RalB protein activity is an allosteric        inhibitor of RalB protein activity;    -   (iii) an inhibitor or depleter of a Src or a Tank Binding Kinase        (TBK1) protein or an inhibitor of Src or TBK1 protein        activation,    -   wherein optionally the inhibitor of the Src or the TBK1 protein        activity is: an amlexanox (or        2-amino-7-isopropyl-5-oxo-5H-chromeno[2,3-b]pyridine-3-carboxylic        acid), or an APHTHASOL™; or    -   a γ(1)34.5 protein of herpes simplex viruses (HSV) (see e.g., Ma        et al., J Virol. 2012 February; 86(4):2188-96); or,    -   a BX795 (as described in, e.g., Bain et al., Biochem J. (2007)        Dec. 15; 408(3):297-315; Clark et al., (2009) J. Biol. Chem.        284:14136-14146); or    -   an azabenzimidazole or an analog or derivative thereof; or    -   a 6-amino-pyrazolopyrimidine or an analog or derivative thereof;        or, a compound having one of the following formulas, or    -   an analog or derivative thereof (see Hutti, et al., (2012)        Development of a High-Throughput Assay for Identifying        Inhibitors of TBK1 and IKKε PLoS ONE 7(7):e41494.doi:        10.1371/journal.pone):

Molecule IKKε TBK1 IKKβ IKKα

0.77 0.44 >10 >10

>10 0.50 >10 >10

>10 0.64 8.76 >10

>10 0.67 >10 >10

>10 0.87 >10 >10

-   -   and optionally the inhibitor of the Src or the TBK1 protein        activity is an allosteric inhibitor of Src or TBK1 protein        activity;    -   (iv) an inhibitor or depleter of a NFKB or a Interferon        regulatory factor 3 (IRF3) protein or an inhibitor of RalB        protein activation,    -   wherein optionally the inhibitor of the NFKB or the IRF3 protein        activity is an allosteric inhibitor of an NFKB or an Interferon        regulatory factor 3 (IRF3) protein activity;    -   (v) an inhibitor or depleter of NFKB or IKK, or an inhibitor of        NFKB or IKK protein activation,    -   wherein optionally the NFKB inhibitor comprises a lenalidomide        or a REVLIMID™ (Celgene Corp., Summit, N.J.) and optionally the        IKK inhibitor comprises a PS1145 (Millennium Pharmaceuticals,        Cambridge, Mass.);    -   (vi) a lenalidomide or a REVLIMID™ and PS1145;    -   (vii) a lenalidomide or a REVLIMID™; a PS1145; and, a Receptor        Tyrosine Kinase (RTK) inhibitor, and optionally the RTK        inhibitor comprises SU14813 (Pfizer, San Diego, Calif.);    -   (viii) an inhibitor of Galectin-3; or    -   (ix) any combination of (i) to (viii), or

(2) one or any combination of the compound, composition or formulation,or compounds, compositions or formulations, of (1), and at least onegrowth factor inhibitor,

wherein optionally the at least one growth factor inhibitor comprises aReceptor Tyrosine Kinase (RTK) inhibitor, a Src inhibitor, ananti-metabolite inhibitor, a gemcitabine, a GEMZAR™, a mitotic poison, apaclitaxel, a taxol, an ABRAXANE™, an erlotinib, a TARCEVA™, alapatinib, a TYKERB™, a cetuxamib, an ERBITUX™, or an insulin growthfactor inhibitor;

wherein optionally the combination or the therapeutic combinationcomprises: (i) an inhibitor or depleter of a Src or a Tank BindingKinase-1 (TBK1) protein or an inhibitor of Src or TBK1 proteinactivation, wherein optionally the inhibitor of the Src or the TBK1protein activity is an amlexanox (or2-amino-7-isopropyl-5-oxo-5H-chromeno[2,3-b]pyridine-3-carboxylic acid)or APHTHASOL™, and (ii) an RTK inhibitor, wherein optionally the RTKinhibitor is a Src inhibitor, an anti-metabolite inhibitor, agemcitabine, a GEMZAR™, a mitotic poison, a paclitaxel, a taxol, anABRAXANE™, an erlotinib, a TARCEVA™, a lapatinib, a TYKERB™, acetuxamib, an ERBITUX™, or an insulin growth factor inhibitor or acombination thereof;

wherein optionally the combination or the therapeutic combinationcomprises an erlotinib with either a Lenalidomide or a PS-1145, or botha Lenalidomide and a PS-1145.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims. All publications,patents, patent applications cited herein are hereby expresslyincorporated by reference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings set forth herein are illustrative of embodiments of theinvention and are not meant to limit the scope of the invention asencompassed by the claims.

FIG. 1 illustrates that integrin αvβ3 expression promotes resistance toEGFR TKI: FIG. 1( a) illustrates flow cytometric quantification of cellsurface markers after 3 weeks treatment with erlotinib (pancreatic andcolon cancer cells) or lapatinib (breast cancer cells); FIG. 1( b)illustrates flow cytometric analysis of αvβ3 expression in FG andMiapaca-2 cells following erlotinib; FIG. 1 (c) illustrates: Top,immunofluorescence staining of integrin αvβ3 in tissue specimensobtained from orthotopic pancreatic tumors treated with vehicle orerlotinib; Bottom, Integrin αvβ3 expression was quantified as ratio ofintegrin αvβ3 pixel area over nuclei pixel area using METAMORPH™; FIG.1( d) Right, intensity of β3 expression in mouse orthotopic lung tumorstreated with vehicle or erlotinib, Left, immunohistochemical staining ofβ3, FIG. 1( e) illustrates data showing that β3 expressing tumor cellswere intrinsically more resistant to EGFR blockade than β3-negativetumor cell lines, where the cells were first screened for αvβ3expression and then analyzed for their sensitivity to EGFR inhibitorserlotinib or lapatinib; FIG. 1( f) illustrates tumor sphere formationassay to establish a dose-response for erlotinib, FIG. 1( g) illustratesorthotopic FG tumors treated for 10 days with vehicle or erlotinib,results are expressed as % tumor weight compared to vehicle control,immunoblot analysis for tumor lysates after 10 days of erlotinibconfirms suppressed EGFR phosphorylation; as discussed in detail inExample 1, below.

FIG. 2 illustrates that integrin αvβ3 cooperates with K-RAS to promoteresistance to EGFR blockade: FIG. 2( a-b) illustrates tumor sphereformation assay of FG tumor cells expressing (a) or lacking (b) integrinβ3 depleted of KRAS (shKRAS) or not (shCTRL) and treated with a doseresponse of erlotinib; FIG. 2( c) illustrates confocal microscopy imagesof PANC-1 and FG-β3 cells grown in suspension; FIG. 2( d) illustrates animmunoblot analysis of RAS activity assay performed in PANC-1 cellsusing GST-Rafl-RBD immunoprecipitation as described below; FIG. 2( e)illustrates an immunoblot analysis of Integrin αvβ3 immunoprecipitatesfrom BxPC-3 β3-positive cells grown in suspension and untreated ortreated with EGF, and RAS activity was determined using a GST-Rafl-RBDimmunoprecipitation assay; as discussed in detail in Example 1, below.

FIG. 3 illustrates that RalB is a key modulator of integrinαvβ3-mediated EGFR TKI resistance: FIG. 3( a) illustrates tumor spheresformation assay of FG-β3 treated with non-silencing (shCTRL) orRalB-specific shRNA and exposed to a dose response of erlotinib; FIG. 3(b) illustrates effects of depletion of RalB on erlotinib sensitivity inβ3-positive tumor in a pancreatic orthotopic tumor model; FIG. 3( c)illustrates tumor spheres formation assay of FG cells ectopicallyexpressing vector control, WT RalB FLAG tagged constructs or aconstitutively active RalB G23V FLAG tagged treated with erlotinib (0.5μM); FIG. 3( d) illustrates RalB activity was determined in FG, FG-β3expressing non-silencing or KRAS-specific shRNA, by using aGST-RalBP1-RBD immunoprecipitation assay; FIG. 3( e) illustrates: Right,overall active Ral immunohistochemical staining intensity between β3negative and β3 positive human tumors; as discussed in detail in Example1, below.

FIG. 4 illustrates that integrin αvβ3/RalB complex leads to NF-μBactivation and resistance to EGFR TKI: FIG. 4( a) illustrates animmunoblot analysis of FG, FG-β3 and FG-β stably expressingnon-silencing or RalB-specific ShRNA, grown in suspension and treatedwith erlotinib (0.5 μM); FIG. 4( b) illustrates tumor spheres formationassay of FG cells ectopically expressing vector control, WT NF-κB FLAGtagged or constitutively active S276D NF-κB FLAG tagged constructstreated with erlotinib; FIG. 4( c) illustrates tumor spheres formationassay of FG-β3 treating with non-silencing (shCTRL) or NF-κB-specificshRNA and exposed to erlotinib; FIG. 4( d) illustrates dose response inFG-β3 cells treated with erlotinib (10 nM to 5 μM), lenalidomide (10 nMto 5 μM) or a combination of erlotinib (10 nM to 5 μM) and lenalidomide(1 μM); FIG. 4( e) illustrates Model depicting the integrinavβ3-mediated EGFR TKI resistance and conquering EGFR™ resistancepathway and its downstream RalB and NF-κB effectors; as discussed indetail in Example 1, below.

FIG. 5 (or Supplementary FIG. 1, Example 1) illustrates that prolongedexposure to erlotinib induces Integrin αvβ3 expression in lung tumors;representative immunohistochemical staining of integrin β3 in mousetissues obtained from H441 orthotopic lung tumors long-term treated witheither vehicle or erlotinib (scale bar, 100 μm); as discussed in detailin Example 1, below.

FIG. 6 (or Supplementary FIG. 2, Example 1) illustrates integrin αvβ3,even in its unligated state, promotes resistance to Growth Factorinhibitors but not to chemotherapies: FIG. 6( a) illustrates a tumorsphere formation assay comparing FG lacking β3 (FG), FG expressing β3wild type (FG-β3) or the β3 D119A (FG-D119A) ligand binding domainmutant, treated with a dose response of erlotinib (Error bars represents.d. (n=3 independent experiments); FIG. 6( b) illustrates tumor sphereformation assay of FG and FG-β3 cells untreated or treated witherlotinib (0.5 μM), OSI-906 (0.1 μM), gemcitabine (0.01 μM) or cisplatin(0.1 μM); FIG. 6( c) illustrates the effect of dose response ofindicated treatments on tumor sphere formation (Error bars represents.d. (n=3 independent experiments); as discussed in detail in Example 1,below.

FIG. 7 (or Supplementary FIG. 3, Example 1) illustrates that integrinαvβ3 does not colocalize with active HRAS, NRAS and RRAS: FIG. 7( a)illustrates that Ras activity was determined in PANC-1 cells grown insuspension by using a GST-Rafl-RBD immunoprecipitation assay asdescribed in Methods, see Example 1 (data are representative of twoindependent experiments); FIG. 7( b) illustrates confocal microscopyimages of PANC-1 cells grown in suspension and stained for KRAS, RRAS,HRAS, NRAS (red), integrin αvβ3 (green) and DNA (TOPRO-3, blue) (Scalebar, 10 μm. Data are representative of two independent experiments); asdiscussed in detail in Example 1, below.

FIG. 8 (or Supplementary FIG. 4, Example 1) illustrates that Galectin-3is required to promote integrin αvβ3/KRAS complex formation: FIG. 8(a-b) illustrates confocal microscopy images of Panc-1 cells lacking orexpressing integrin αvβ3 grown in suspension; FIG. 8( a) illustratescells stained for KRAS (green), Galectin-3 (red), and DNA (TOPRO-3,blue); FIG. 8( b) illustrates cells stained for integrin αvβ3 (green),Galectin-3 (red) and DNA (TOPRO-3, blue), Scale bar, 10 μm, data arerepresentative of three independent experiments; FIG. 8( c) illustratesan immunoblot analysis of Galectin-3 immuno-precipitates from PANC-1cells expressing non-silencing (sh CTRL) or integrin β3-specific shRNA(sh β3), data are representative of three independent experiments; FIG.8( d) illustrates an immunoblot analysis of integrin β3immunoprecipitates from PANC-1 cells expressing non-silencing (sh CTRL)or Galectin-3-specific shRNA (sh Ga13), data are representative of threeindependent experiments; as discussed in detail in Example 1, below.

FIG. 9 (or Supplementary FIG. 5, Example 1) illustrates that ERK, AKTand RalA are not specifically required to promote integrin αvβ3-mediatedresistance to EGFR™; FIG. 9A β3-negative cells, and FIG. 9B, β3-positivecells; tumor spheres formation assay of FG and FG-β3 expressingnon-silencing or ERK1/2, AKT1 and RalA-specific shRNA and treated witherlotinib (0.5 μM), error bars represent s.d. (n=3 independentexperiments); as discussed in detail in Example 1, below.

FIG. 10 (or Supplementary FIG. 6, Example 1) illustrates that RalB issufficient to promote resistance to EGFR TKI: FIG. 10( a) (supplementaryFIG. 6, Example 1) illustrates a tumor sphere formation assay of FGexpressing non-silencing or RalB specific shRNA and treated with a doseresponse of erlotinib. Error bars represent s.d. (n=3 independentexperiments); FIG. 10( b) (supplementary FIG. 6) illustrates a tumorspheres formation assay of PANC-1 stably expressing integrin β3-specificshRNA and ectopically expressing vector control, WT RalB FLAG tagged ora constitutively active RalB G23V FLAG tagged constructs treated witherlotinib (0.5 μM), error bars represent s.d. (n=3 independentexperiments); FIG. 10( c) (Supplementary FIG. 7, Example 1) shows thatintegrin αvβ3 colocalizes with RalB in cancer cells: illustratesconfocal microscopy images of Panc-1 cells grown in suspension. Cellsare stained for integrin αvβ3 (green), RalB (red), pFAK (red), and DNA(TOPRO-3, blue), scale bar, 10 μm, data are representative of threeindependent experiments; as discussed in detail in Example 1, below.

FIG. 11 (or Supplementary FIG. 8, Example 1) illustrates that integrinαvβ3 colocalizes with RalB in human breast and pancreatic tumor biopsiesand interacts with RalB in cancer cells: FIG. 11( a) illustratesconfocal microscopy images of integrin αvβ3 (green), RalB (red) and DNA(TOPRO-3, blue) in tumor biopsies from breast and pancreatic cancerpatients, Scale bar, 20 μm; FIG. 11( b) illustrates a Ral activity assayperformed in PANC-1 cells using GST-RalBP1-RBD immunoprecipitationassay, Immunoblot analysis of RalB and integrin β, data arerepresentative of three independent experiments; as discussed in detailin Example 1, below.

FIG. 12 (or FIG. 1 in Example 2) illustrates data showing that integrinβ3 is expressed in EGFR inhibitor resistant tumors and is necessary andsufficient to drive EGFR inhibitor resistance: FIG. 12(A) schematicallyillustrates that the identification of the most upregulated tumorprogression genes common to erlotinib resistant carcinomas; FIG. 12(B)in table form shows Erlotinib IC₅₀ in a panel of human carcinoma celllines treated with erlotinib in 3D culture; FIG. 12(C) graphicallyillustrates percentage of integrin β3 positive cells in parental linesvs. after 3 or 8 weeks treatment with erlotinib; FIG. 12(D) graphicallyillustrates quantification of integrin β3 (ITGβ3) gene expression inhuman lung cancer biopsies from patients from the BATTLE Study (18) whowere previously treated with an EGFR inhibitor and progressed (n=27),versus patients who were EGFR inhibitor naïve (n=39); FIG. 12(E)illustrates images of paired human lung cancer biopsies obtained beforeand after erlotinib resistance were immunohistochemically stained forintegrin β, scale bar, 50 μm; FIG. 12(F) graphically illustrates: Rightgraph shows effect of integrin β3 knockdown on erlotinib resistance ofβ-positive cells, and Left graph shows effect of integrin β3 ectopicexpression on erlotinib resistance in FG and H441 cells; FIG. 12(G)graphically illustrates: Right graph shows the effect of integrin β3knockdown on erlotinib resistance in vivo, A549 shCTRL and A549 shintegrin β3 (n=8 per treatment group) were treated with erlotinib (25mg/kg/day) or vehicle during 16 days, results are expressed as averageof tumor volume at day 16. *P<0.05; and Left graph shows orthotopic FGand FG-β3 tumors treated for 30 days with vehicle or erlotinib, resultsare expressed as % tumor weight compared to vehicle control; as furtherdescribed in Example 2, below.

FIG. 13 (or FIG. 2 in Example 2) illustrates data showing that integrinβ3 is required to promote KRAS dependency and KRAS-mediated EGFRinhibitor resistance: FIG. 13(A) illustrates confocal microscopy imagesshowing immunostaining for integrin β3 (green), K-, N-, H-, R-Ras (red),and DNA (TOPRO-3, blue) for BxPc3 cells grown in suspension in mediawith 10% serum, arrows indicate clusters where integrin β3 and KRAScolocalize (yellow); FIG. 13(B-C) illustrates confocal microscopy imagesshowing immunostaining for integrin β3 (green), KRAs (red) and DNA(Topro-3, blue) for PANC-1 (KRAS mutant) and HCC827 (KRAS wild-type)after acquired resistance to erlotinib (HCC827R) grown in suspension inabsence (Vehicle) or in presence of erlotinib (0.5 μM and 0.1 μMrespectively), arrows indicate clusters where integrin β3 and KRAScolocalize (yellow); FIG. 13(D) graphically illustrates the effect ofKRAS knockdown on tumorspheres formation in a panel of lung andpancreatic cancer cells expressing or lacking integrin β3; FIG. 13(E)graphically illustrates the effect of KRAS knockdown on tumorsphereformation in PANC-1 (KRAS mutant) stably expressing non-target shRNAcontrol (0-positive) or specific-integrin β3 shRNA (β3 negative) in FG(KRAS mutant) and BxPc3 (KRAS wild-type) stably expressing vectorcontrol or integrin β3; FIG. 13(F) graphically illustrates the effect ofKRAS knockdown on erlotinib resistance of β3-negative and β3-positiveepithelial cancer cell lines, cells were treated with a dose response oferlotinib; FIG. 13(G) illustrates confocal microscopy images showingimmunostaining for integrin β3 (green), KRAS (red) and DNA (TOPRO-3,blue) for PANC-1 cells expressing non-target shRNA control or Galectin3-specific shRNA grown in suspension; FIG. 13(H) illustrates: Top:immunoblot analysis of integrin β3 immunoprecipitates from PANC-1 cellsexpressing non-target shRNA control (CTRL) or Galectin-3-specific shRNA(Gal-3); Bottom: immunoblot analysis of Galectin-3 immunoprecipitatesfrom PANC-1 cells expressing non-target shRNA control (CTRL) or integrinβ3-specific shRNA (β3); FIG. 13(I) graphically illustrates erlotinibdose response of FG-β3 cells expressing a non-target shRNA control or aGalectin-3-specific shRNA (sh Gal-3); as further described in Example 2,below.

FIG. 14 (or FIG. 3 in Example 2) illustrates data showing that RalB is acentral player of integrin β3-mediated EGFR inhibitor resistance: FIG.14(A) graphically illustrates the effect of RalB knockdown on erlotinibresistance of β3-positive epithelial cancer cell lines, cells weretreated with 0.5 μM of erlotinib: FIG. 14(B) graphically illustrates theeffect of RalB knockdown on erlotinib resistance of β3-positive humanpancreatic (FG-β3) orthotopic tumor xenografts, established tumorsexpressing non-target shRNA, (shCTRL) or a shRNA targeting RalB (shRalB) were randomized and treated for 10 days with vehicle or erlotinib,results are expressed as % of tumor weight changes after erlotinibtreatment compared to vehicle; FIG. 14(C) graphically illustrates theeffect of expression of a constitutively active Ral G23V mutant onerlotinib response of β3 negative cells, cells were treated with 0.5 μMof erlotinib; FIG. 14(D) illustrates the effect of expression ofintegrin β3 on KRAS and RalB membrane localization; FIG. 14(E)illustrates Ral activity that was determined in PANC-1 cells grown insuspension by using a GST-RalBP1-RBD immunoprecipitation assay,immunoblots indicate RalB activity and association of active RalB withintegrin β3; FIG. 14(F) illustrates confocal microscopy images ofintegrin αvβ3 (green), RalB (red) and DNA (TOPRO-3, blue) in tumorbiopsies from pancreatic cancer patients; FIG. 14(G) illustrates theeffect of β3 expression and KRAS expression on RalB activity, measuredusing a GST-RalBP1-RBD immunoprecipitation assay; FIG. 14(H) illustratesimmunoblot analysis of FG and FG-β3 stably expressing non-target shRNAcontrol or RalB-specific shRNA, grown in suspension and treated witherlotinib (0.5 μM); FIG. 14(I) graphically illustrates the effect of aTank Binding Kinase (TBK1) and p65 NFκB on erlotinib resistance of FG-β3cells, cells were treated with 0.5 μM of erlotinib; as further describedin Example 2, below.

FIG. 15 (or FIG. 4 in Example 2) illustrates data showing that reversalof β3-mediated EGFR inhibitor resistance in oncogenic KRAS model bypharmacological inhibition: FIG. 15(A) graphically illustrates theeffect of NFkB inhibitors on erlotinib response of β3-positive cells(FG-β3, PANC-1 and A549), cells were treated with vehicle, erlotinib(0.5 μM), lenalidomide (1-2 μM), bortezomib (4 nM) alone or incombination; FIG. 15(B) graphically illustrates data from: Left, micebearing subcutaneous β3-positive tumors (FG-β3) were treated withvehicle, erlotinib (25 mg/kg/day), lenalidomide (25 mg/kg/day) or thecombination of erlotinib and lenalidomide, tumor dimensions are reportedas the fold change relative to size of the same tumor on Day 1; Right,mice bearing subcutaneous β3-positive tumors (FG-R) after acquiredresistance to erlotinib were treated with vehicle, erlotinib (25mg/kg/day), bortezomib (0.25 mg/kg), the combination of erlotinib andbortezomib, tumor dimensions are reported as the fold change relative tosize of the same tumor on Day 1; FIG. 15(C) schematically illustrates amodel depicting an integrin αvβ3-mediated KRAS dependency and EGFRinhibitor resistance mechanism; as further described in Example 2,below.

FIG. 16 (or supplementary FIG. S1, in Example 2) illustrates datashowing that illustrates resistance to EGFR inhibitor is associated withintegrin β3 expression in pancreatic and lung human carcinoma celllines: FIG. 16(A) illustrates immunoblots showing integrin β3 expressionin human cell lines used in FIG. 12; FIG. 16(B) graphically illustratesdata showing the effect of erlotinib on HCC827 xenograft tumors inimmuno—compromised mice relative to vehicle-treated control tumors; FIG.16(C) left, graphically illustrates data of Integrin αvβ3 quantificationin orthotopic lung (upper panel) and pancreas (lower panel) tumorstreated with vehicle or erlotinib until resistance, FIG. 16(C) right,illustrates a representative immunofluorescent staining of integrin αvβ3in lung (upper panel) and pancreatic (lower panel) human xenograftstreated 4 weeks with vehicle or erlotinib; as further described inExample 2, below.

FIG. 17 (or supplementary FIG. S2, in Example 2) illustrates Integrin β3expression predicts intrinsic resistance to EGFR inhibitors in tumors;FIG. 17A graphically illustrates a plot of progression-free survival forerlotinib-treated patients with low versus (vs.) high protein expressionof β3 integrin measured from non-small cell lung cancer biopsy material(FIG. 17B illustrates: in right panel β3 integrin high cells and leftpanel β3 integrin low cells) obtained at diagnosis; as further describedin Example 2, below.

FIG. 18 (or supplementary FIG. S3, in Example 2) illustrates Integrin β3confers Receptor Tyrosine Kinase inhibitor resistance: FIG. 18(A)illustrates immunoblots showing integrin β3 knockdown efficiency incells used in FIG. 12; FIG. 18(B) graphically illustrates response ofA549 lung carcinoma cells non-target shRNA control or shRNA targetingintegrin β3 to treatment with either vehicle or erlotinib (25 mg/kg/day)during 16 days; FIG. 18(C) illustrates immunoblots showing expression ofindicated proteins of representative tumors; FIG. 18(D) illustratesrepresentative photographs of crystal violet-stained tumorspheres ofβ3-negative and β3-positive cells after erlotinib, OSI-906, gemcitabineand cisplatin treatment; FIG. 18(E) graphically illustrates the effectof integrin β3 expression on lapatinib and OSI-906 (left panel), andcisplatin and gemcitabine (right panel); FIG. 18(F) graphicallyillustrates data from a viability assay of FG and FG-β3 cells grown insuspension in media with or without serum; as further described inExample 2, below.

FIG. 19 (or supplementary FIG. S4, in Example 2) illustrates integrinβ3-mediated EGFR inhibitor resistance is independent of its ligandbinding: FIG. 19A graphically illustrates the effect of ectopicexpression of β3 wild-type (FG-β3) or the β3 D119A (FG-D119A) ligandbinding domain mutant on erlotinib response; FIG. 19B illustrates animmunoblot showing transfection efficiency of vector control, integrinβ3 wild-type and integrin β3 D119A; as further described in Example 2,below.

FIG. 20 (or supplementary FIG. S5, in Example 2) illustrates integrin β3colocalizes and interacts with oncogenic and active wild-type KRAS: FIG.20(A) illustrates confocal microscopy images of FG and FG-β3 cells grownin suspension in media 10% serum with or without erlotinib (0.5 μM) andstained for KRAS (red), integrin αvβ3 (green) and DNA (TOPRO-3, blue);FIG. 20(B) illustrates Ras activity was determined in PANC-1 cells grownin suspension by using a GST-Rafl-RBD immunoprecipitation assay,immunoblots indicate KRAS activity and association of active KRAS withintegrin β3; FIG. 20(C) illustrates an immunoblot analysis showing thatIntegrin αvβ3 immunoprecipitates from BxPC-3 cells grown in suspensionin presence or absence of growth factors; as further described inExample 2, below.

FIG. 21 (or supplementary FIG. S6, in Example 2) illustrates integrin β3expression promotes KRAS dependency: FIG. 21(A) illustrates Immunoblotsshowing KRAS knockdown efficiency in cells used in FIG. 13; FIG. 21(B)illustrates Representative photographs of crystal violet-stainedtumorspheres of FG and A549 cells expressing non-target shRNA control orspecific-KRAS shRNA; FIG. 21(C) illustrates the effect of an additionalKRAS knockdown on tumorspheres formation in PANC-1 stably expressingnon-target shRNA control (β3-positive) or specific-integrin β3 shRNA (β3negative); FIG. 21(D) illustrates immunoblots showing KRAS knockdownefficiency; as further described in Example 2, below.

FIG. 22 (or supplementary FIG. S7, in Example 2) illustrates imagesshowing that KRAS and Galectin-3 colocalize in integrin β3-positivecells, in particular, confocal microscopy images of FG and FG-β3 cellsgrown in suspension and stained for KRAS (green), galectin-3 (red) andDNA (TOPRO-3, blue); as further described in Example 2, below.

FIG. 23 (or supplementary FIG. S8, in Example 2) illustrates Integrinβ3-mediated KRAS dependency and erlotinib resistance is independent ofERK, AKT and RalA: FIG. 23(A) graphically illustrates the effect of ERK,AKT, RalA and RalB knockdown on erlotinib response (erlotinib 0.5 μM) ofβ3-negative FG (left panel) and β3-positive FG-β3 cells (right panel);FIG. 23(B) illustrates Immunoblots showing ERK, AKT RalA and RalBknockdown efficiency on β3-negative FG (upper panel) and β3-positiveFG-β3 cells (lower panel); FIG. 23(C) illustrates Immunoblots showingRalB knockdown efficiency in the β3-positive epithelial cancer cellsused in FIG. 14; as further described in Example 2, below.

FIG. 24 (or supplementary Figure S9, in Example 2) illustratesconstitutive active NFkB is sufficient to promote erlotinib resistance:FIG. 24(A) illustrates immunoblots showing a Tank Binding Kinase (TBK1)(upper panel) and NFkB knockdown efficiency (lower panel) used in FIG.14; FIG. 24(B) graphically illustrates the effect of constitutive activeS276D p65NFkB on erlotinib response (erlotinib 0.5 OA) of β3-negativecells (FG cells); as further described in Example 2, below.

FIG. 25 (or supplementary Figure S10, in Example 2) illustrates NFkBinhibitors in combination with erlotinib increase cell death in vivo:FIG. 25(A) and FIG. 25 (B) illustrate Immunoblots showing expression ofindicated proteins of representative tumors from shown in FIG. 15B; FIG.25(C) illustrates Confocal microscopy images of cleaved caspase 3 (red)and DNA (TOPRO-3, blue) in tumor biopsies from xenografts tumors used inFIG. 15B treated with vehicle, erlotinib, lenalidomide or lenalidomideand erlotinib in combo; FIG. 25(D) illustrates Confocal microscopyimages of cleaved caspase 3 (red) and DNA (TOPRO-3, blue) in tumorbiopsies from xenografts tumors used in FIG. 15B treated with vehicle,erlotinib, bortezomib or bortezomib and erlotinib in combo); as furtherdescribed in Example 2, below.

FIGS. 26, 27, and 28, illustrate supplementary Table 1 from Example 2,showing that differentially expressed genes in cells resistant toerlotinib (PANC-1, H1650, A459) compared with the average of twosensitive cells (FG, H441) and in HCC827 after acquired resistance invivo (HCC827R) vs. the HCC827 vehicle-treated control; as furtherdescribed in Example 2, below.

FIG. 29 illustrates supplementary Table 2, from Example 2, showing KRASmutational status in pancreatic and lung cell lines used in the study ofExample 2, below.

FIG. 30 illustrates data showing integrin β3 (CD61) is a RTKI (ReceptorTyrosine Kinase (RTK) Inhibitor) drug resistance biomarker on thesurface of circulating tumor cells; as discussed in detail in Example 2,below. As schematically illustrated in FIG. 30A, CD61 (β3, or beta3)negative human lung cancer cells (HCC827; this lung adenocarcinoma hasan acquired mutation in the EGFR tyrosine kinase domain (E746-A750deletion), and they are sensitive to erlotinib and develop acquiredresistance after 6/8 weeks) were injected orthotopically into the lungof mice and treated over 3 months with erotinib at 25 mg/kg/day. Asgraphically illustrated in FIG. 30B, Human lung cancer cells detected inthe circulation were positive for αvβ3 (or avb3, CD61) whereas the cellsin the untreated group were essentially negative for this marker. CD45negative cells indicates that the detected cells were not leukocytes andpan cytokeratin positive cells indicate tumor cells. CD61 (beta3)positive expression correlated with tumor expression.

FIG. 31 illustrates data showing how targeting the NF-κB pathway usingcompositions and methods of this invention can sensitize resistanttumors to growth factor inhibitors by showing the effect of NFkBinhibitors on erlotinib response of β3-negative (b3-negative) cells (FG)and β3-positive cells (FG-β3, MDA-MB231 (intrinsic resistance, FIG. 31A)and FG-R (acquired resistance, FIG. 31B), and EGFR TKI (Tyrosine KinaseInhibitor) sensitive cells, FIG. 31C. Cells embedded in agar (anchorageindependent growth) were treated with vehicle, erlotinib (0.5 μM),Lenalidomide (2 μM), PS-1145 (1 μM) alone or in combination for 10 to 15days. Then, the soft agar were stained with crystal violet and thecolonies were counted manually. The results show that while β3-positivecells (intrinsic FIG. 31A or acquired resistant FIG. 31B cells) wereresistant to erlotinib and each NFκB inhibitor alone, the combination oferlotinib with either Lenalidomide or PS-1145 decreased tumorsphereformation.

FIG. 32 (or FIG. 1 of Example 3) illustrates: Integrin β3 expressionincrease tumor-initiating and self-renewal capacities: FIG. 32( a)Limiting dilution in vivo determining the frequency of tumor-initiatingcells for A549 cells expressing non-target shRNA control or integrinβ3-specific shRNA and for FG cells expressing control vector or integrinβ3 (FG-β3); FIG. 32( b-c-d) Self-renewal capacity of A549 (FIG. 32 b)and PANC-1 (FIG. 32 c) cells expressing non-target shRNA control (CTRL)or integrin specific shRNA and of FG expressing control vector orintegrin β3 (FG-β3) (FIG. 32 d); as described in detail in Example 3,below.

FIG. 33 (or FIG. 2, of Example 3) illustrates: Integrin β3 drivesresistance to EGFR inhibitors: FIG. 33( a) graphically illustrates theEffect of integrin β3 expression (ectopic expression for FG and integrinβ3-specific knockdown for PANC-1) cells on drug treatment response; FIG.33( b) graphically illustrates the Effect of integrin β3 knockdown onerlotinib response in MDA-MB-231 (MDA231), A549 and H1650; FIGS. 33( c)and 33(d) graphically illustrate the effect of integrin β3 knockdown onerlotinib resistance in vivo using A549 shCTRL and A549 sh β3 treatedwith erlotinib or vehicle, FIG. 33( c) measuring tumorspheres, and β3(d)measuring tumor volume in A549 shCTRL (integrin β3+), left panel, andA549 (integrin β3−) (right panel); FIG. 33( e) [[33(d)]] graphicallyillustrates Orthotopic FG and FG-β3 tumors (>1000 mm³; n=5 per treatmentgroup) were treated for 30 days with vehicle or erlotinib; FIG. 33( f)graphically illustrates Relative mRNA expression of integrin β3 (ITGB3)in HCC827 vehicle-treated tumors (n=5) or erlotinib-treated tumors (n=7)from (e) after acquired resistance; FIG. 33( g) H&E sections andimmunohistochemical analysis of integrin β3 expression in paired humanlung cancer biopsies obtained before and after erlotinib resistance;FIG. 33( h) illustrates images of Limiting dilution in vivo determiningthe frequency of tumor-initiating cells for HCC827 vehicle-treated(vehicle) and erlotinib-treated tumors from (erlotinib resistantnon-sorted) (e); FIG. 33( i) and FIG. 33( j) graphically illustrate theSelf-renewal capacity of HCC827 vehicle-treated (vehicle),erlotinib-treated (erlotinib resistant non-sorted), erlotinib-treatedintegrin β3-population and erlotinib-treated integrin β3+ population; asdescribed in detail in Example 3, below.

FIG. 34 (or FIG. 3, of Example 3) illustrates: Integrin β3/KRAS complexis critical for integrin β3-mediated stemness: FIG. 34( a) Confocalmicroscopy images show immunostaining for Integrin β3 (green), KRAS(red) and DNA (TOPRO-3, blue) for FG-β3, A549 and HCC827 after acquiredresistance to erlotinib (HCC827 ER) grown in suspension, Arrows indicateclusters where integrin β3 and KRAS colocalize (yellow); FIG. 34( b) Rasactivity was determined in PANC-1 cells grown in suspension by using aGST-Rafl-RBD immunoprecipitation assay, Immunoblots indicate KRASactivity and association of active KRAS with integrin β3; FIG. 34( c)Effect of KRAS knockdown on tumorspheres formation in lung (A549 andH441) and pancreatic (FG and PANC-1) cancer cells expressing or lackingintegrin β3; FIG. 34( d) Effect of KRAS knockdown on erlotinibresistance of β3-negative and β3-positive epithelial cancer cell lines,Cells were treated with a dose response of erlotinib; FIG. 34( e)Self-renewal capacity of FG-β3 cells expressing non-target shRNA control(shCTRL) or KRAS-specific shRNA measured by quantifying the number ofprimary and secondary tumorspheres; FIG. 34( f) Confocal microscopyimages show immunostaining for integrin β3 (green), KRAS (red) and DNA(TOPRO-3, blue) for PANC-1 cells expressing non-target shRNA control orGalectin 3-specific shRNA grown in suspension; FIG. 34( g) immunoblotanalysis of integrin β3 immunoprecipitates from PANC-1 cells expressingnon-target shRNA control (CTRL) or Galectin-3-specific shRNA (Gal-3);FIG. 34( h) Effect of Galectin-3 knockdown on integrin β3-mediatedanchorage independent growth and erlotinib resistance; FIG. 34( i)Self-renewal capacity of PANC-1 cells expressing non-target shRNAcontrol (shCTRL) or Galectin-3-specific shRNA (sh Gal-3) measured byquantifying the number of primary and secondary tumorspheres; asdescribed in detail in Example 3, below.

FIG. 35 (or FIG. 4, of Example 3) illustrates: RalB/TBK1 signaling is akey modulator of integrin ⊖3-mediated stemness: FIG. 35( a) Effect ofRalB knockdown on anchorage independence; FIG. 35( b) Self-renewalcapacity of FG-β3 cells expressing non-target shRNA control (sh CTRL) orRalB-specific shRNA (sh RalB) measured by quantifying the number ofprimary and secondary tumorspheres; FIG. 35( c) Limiting dilution invivo determining the frequency of tumor-initiating cells for FG-β3 cellsexpressing non-target shRNA control or integrin RalB-specific shRNA;FIG. 35( d) Effect of RalB knockdown on erlotinib resistance ofβ3-positive epithelial cancer cell lines; FIG. 35( e) Effect of RalBknockdown on erlotinib resistance of β3-positive human pancreatic(FG-β3) orthotopic tumor xenografts. Established tumors expressingnon-target shRNA, (sh CTRL) or a shRNA targeting RalB (sh RalB); FIG.35( f) Immunoblot analysis of FG and FG-β3 stably expressing non-targetshRNA control or RalB-specific shRNA, grown in 3D and treated witherlotinib (0.5 μM); FIG. 35( g) Effect of TBK1 knockdown on PANC-1self-renewal capacity; FIG. 35( h) Effect of TBK1 knockdown on erlotinibresistance of PANC-1 cells. Cells were treated with 0.5 μM of erlotinib;FIG. 35( i) Mice bearing subcutaneous β3-positive tumors (PANC-1) weretreated with vehicle, erlotinib (25 mg/kg/day), amlexanox (25 mg/kg/day)or the combination of erlotinib and amlexanox; as described in detail inExample 3, below.

FIG. 36 (or Figure S1, of Example 3) illustrates: FIG. 36( a-b) Limitingdilution tables; FIG. 36( c) Immunoblots showing integrin β3 knockdownor ectopic expression efficiency in cells used in FIG. 1 (of Example 3);FIG. 36( d) Viability assay (CellTiter-Glo assay) of FG and FG-β3 cellsgrown in 3D in media with or without serum; FIG. 36( e)Immunohistochemical analysis of integrin β3 expression in paired humanlung cancer biopsies obtained before (upper panel) and after (lowerpanel) erlotinib resistance; FIG. 36( f) Limiting dilution table; FIG.36( g) image of Immunohistochemistry staining of CD166 (upper panel) andintegrin β3 (lower panel) in human lung tumor biopsies after EGFR™acquired resistance; as described in detail in Example 3, below.

FIG. 37 (or Figure S2, of Example 3) illustrates: FIG. 37( a) Effect ofcilengetide treatment on erlotinib resistance in FG-β3 and PANC-1 cells;FIG. 37( b) Effect of ectopic expression of β3 wild-type (FG-β3) or theβ3 D119A (FG-D119A) ligand binding domain mutant on erlotinib response;FIG. 37( c) Confocal microscopy images of FG-β3 cells grown in 3D andstained for integrin—β3 (green) and RAS family members (red); FIG. 37(d) Immunoblots showing KRAS knockdown efficiency in cells used in FIG. 3(of Example 3); FIG. 37( e) Representative photographs of crystalviolet-stained tumorspheres of FG and A549 cells expressing non-targetshRNA control or specific-KRAS; FIG. 37( f) illustrates the Effect of asecond KRAS knockdown (shKRAS 2) on tumorspheres formation in PANC-1stably expressing non-target shRNA control (3-positive) orspecific-integrin-β3 shRNA (3 negative), left panel graphicallypresenting data and right panel illustrating an immunoblot showing KRASexpression in sh CTRL, SH KRAS and sh KRAS 2; as described in detail inExample 3, below.

FIG. 38 (or Figure S3, of Example 3) illustrates: FIG. 38( a)graphically illustrates the Effect of ERK, AKT and RalA knockdown onerlotinib response of β3-negative FG and 3-positive FG-3 cells; FIG. 38(b) Immunoblots showing ERK, AKT and RalA knockdown efficiency in cellsused in (a); FIG. 38( c) Immunoblots showing RalB knockdown efficiencyin cells used in FIG. 3 (of Example 3); FIG. 38( d) graphicallyillustrates the effect of a second RalB knockdown (shRalB 2) ontumorspheres formation in PANC-1 stably expressing non-target shRNAcontrol (β3-positive) or specific-integrin β3 shRNA (3 negative); FIG.38( e) Limiting dilution table; FIG. 38( f) Confocal microscopy imagesof integrin αvβ3 (green), RalB (red) and DNA (TOPRO-3, blue) in tumorbiopsies from pancreatic cancer patients; FIG. 38( g) Ral activity wasdetermined in PANC-1 cells grown in suspension by using a GST-RalBP1-RBDimmunoprecipitation assay. Immunoblots indicate RalA and RalBactivities; FIG. 38( h) Effect of β3 expression and KRAS expression onRalB activity, measured using a GST-RalBP1-RBD immunoprecipitationassay; FIG. 38( i) illustrates the effect of expression of aconstitutively active Ral G23V mutant on erlotinib resistance of β3positive and negative cells, left panel graphically presenting data andright panel illustrating an immunoblot showing FLAG, RalB and Hsp90expression; as described in detail in Example 3, below.

FIG. 39 (or Figure S4, of Example 3) illustrates: FIG. 39( a) Immunoblotshowing TBK1 knockdown efficiency in PANC-1 cells used in FIG. 4 (ofExample 3); FIG. 39( b) Effect of the TBK1 inhibitor amlexanox onerlotinib response of PANC-1 cells; FIG. 39( c) Effect of the NFkBinhibitor borthezomib on β3-positive cells (FG-β3 (left panel), PANC-1(middle panel) and A549 (right panel)); FIG. 39( d) Mice bearingsubcutaneous β3-positive tumors (FG-β3) were treated with vehicle,erlotinib (25 mg/kg/day), bortezomib (0.25 mg/kg), the combination oferlotinib and bortezomib; FIG. 39( e) Confocal microscopy images ofcleaved caspase 3 (red) and DNA (TOPRO-3, blue) in tumor biopsies fromxenografts tumors used in (d) treated with vehicle, erlotinib,bortezomib or bortezomib and erlotinib in combo; as described in detailin Example 3, below.

FIG. 40 graphically illustrates data demonstrating that depletion ofRalB overcomes erlotinib resistance in KRAS mutant cells: FIG. 40Agraphically illustrates number of tumorspheres as a percent of controlfor FG, FG-beta3, PANC-1, and A539 expressing cells, with or withouterlotinib, in vitro soft agar conditions; and FIG. 40B graphicallyillustrates tumor weight as a percent of control, in in vivo orthotopicpancreas xenograft; as discussed in detail in Example 2, below.

FIG. 41 graphically illustrates data demonstrating that depletion ofTBK1 overcomes erlotinib resistance in KRAS mutant cells: FIG. 41Aillustrates data demonstrating that integrin mediates TBK1 activationthrough Ral b; FIG. 41B and FIG. 41C graphically illustrate datademonstrating TBK1 depletion (with siRNA) overcomes integrinbeta-3-mediated erlotinib resistance, where FIG. 41A shows the number oftumorspheres as a percent of non-treated cells with and without siRNAdepletion of TBK1, and FIG. 41C shows tumor size as a percent of controlwith erlotinib, amlexanox and erlotinib+amlexanox; as discussed indetail in Example 2, below.

Like reference symbols in the various drawings indicate like elements.

Reference will now be made in detail to various exemplary embodiments ofthe invention, examples of which are illustrated in the accompanyingdrawings. The following detailed description is provided to give thereader a better understanding of certain details of aspects andembodiments of the invention, and should not be interpreted as alimitation on the scope of the invention.

DETAILED DESCRIPTION

In alternative embodiments, the invention provides compositions andmethods for overcoming or diminishing or preventing Growth FactorInhibitor (GFI) resistance in a cell, or, a method for increasing thegrowth-inhibiting effectiveness of a Growth Factor inhibitor on a cell,or, a method for re-sensitizing a cell to a Growth Factor Inhibitor(GFI). In alternative embodiments, the cell is a tumor cell, a cancercell or a dysfunctional cell. In alternative embodiments, the inventionprovides compositions and methods for determining: whether an individualor a patient would benefit from or respond to administration of a GrowthFactor Inhibitor, or, which individuals or patients would benefit from acombinatorial approach comprising administration of a combination of: atleast one growth factor and at least one compound, composition orformulation used to practice a method of the invention, such as an NfKbinhibitor.

We found that integrin anb3 is upregulated in cells that becomeresistant to Growth Factor inhibitors. Our findings demonstrate thatintegrin anb3 promotes de novo and acquired resistance to Growth factorinhibitors by interacting and activating RalB. RalB activation leads tothe activation of Src and TBK1 and the downstream effectors NFKB andIRF3. We also found that depletion of RalB or its downstream signaling(Src/NFKB) in b3-positive cells overcomes resistance to growth factorinhibitors. This invention demonstrates that the integrin anb3/RalBsignaling complex promotes resistance to growth factor inhibitors; andin alternative embodiments, integrin α_(v)β₃ (anb3) and active RalB areused as biomarkers in patient samples to predict which patients willrespond to growth factor inhibitors and which patients might ratherbenefit from alternative/combinatorial approaches such as a combinationof growth factor inhibitors and NfKb inhibitors.

This invention for the first time identifies integrin αvβ3 and activeRalB as potential biomarker for tumors that are or have become (e.g., denovo and acquired) resistant to growth factors blockade. Accordingly, inalternative embodiments, the invention provides compositions and methodsfor the depletion of RalB, Src, NFkB and its downstream signalingeffectors to sensitize αvβ3-expressing tumors to growth factor blockade.These findings reveal a new role for integrin αvβ3 in mediating tumorcell resistance to growth factor inhibition and demonstrate thattargeting the αvβ3/RalB/10 NfkB/Src signaling pathway will circumventgrowth factor resistance of a wide range of cancers.

In alternative embodiments, any NF-kB inhibitor can be used to practicethis invention, e.g., lenalidomide or(RS)-3-(4-amino-1-oxo-3H-isoindol-2-yl)piperidine-2,6-dione, which canbe REVLIMID™ (Celgene Corp., Summit, N.J.), or thalidomide, or any otherderivative of thalidomide, or any composition having an equivalentactivity.

In alternative embodiments, compositions and methods of the inventionare used to sensitize tumors to drugs, e.g., such as erlotinib andlapatinib (which are commonly used to treat a wide range of solidtumors). We have shown that when tumors become resistant to these drugsthey become very sensitive to NFkB inhibitors. Thus, in alternativeembodiments, compositions and methods of the invention are used tosensitize tumors using NFkB inhibitors, such as e.g., lenalidomide or(RS)-3-(4-amino-1-oxo-3H-isoindol-2-yl)piperidine-2,6-dione orREVLIMID™, or a composition as listed in Table 1.

In alternative embodiments, compositions and methods of the inventionare used to sensitize tumors using an IKK inhibitor, e.g., such asPS1145 (Millennium Pharmaceuticals, Cambridge, Mass.) (see e.g.,Khanbolooki, et al., Mol Cancer Ther 2006; vol. 5:2251-2260; Publishedonline Sep. 19, 2006; Yemelyanov, et al., Oncogene (2006) vol.25:387-398; published online 19 Sep. 2005), or any IKBα (nuclear factorof kappa light polypeptide gene enhancer in B-cells inhibitor, alpha)phosphorylation and/or degradation inhibitor, e.g., one or morecompositions listed in Table 3.

In alternative embodiments, compositions and methods of the inventioncomprise use of an NFkB inhibitor and an IKK inhibitor to treat a drugresistant tumor, e.g., a solid tumor. In alternative embodiments,compositions and methods of the invention comprise use of an NFkBinhibitor and an IKK inhibitor to treat a drug resistant tumor incombination with an anticancer drug, e.g., an NFkB inhibitor and an IKKinhibitor are used to sensitize a tumor to drugs such as erlotinib andlapatinib. In alternative embodiments, the drug combination used topractice the invention comprises lenalidomide (such as a REVLIMID™) andthe IKK inhibitor PS1145 (Millennium Pharmaceuticals, Cambridge, Mass.).For example, lenalidomide (such as a REVLIMID™) and PS1145 are used tosensitize a tumor that is resistant to a cancer drug, e.g., an EGFRinhibitor, such that the tumor is now responsive to the cancer drug.

In alternative embodiments, in practicing the invention, an NFkBinhibitor and an IKK inhibitor are used in combination with a tyrosinekinase receptor (also called Receptor Tyrosine Kinases, or RTKs)inhibitor, e.g., an SU14813 (Pfizer, San Diego, Calif.) or as listed inTable 2 or 3, below, to treat a drug resistant tumor. In alternativeembodiments, compositions and methods of the invention (e.g., includinglenalidomide or PS1145; lenalidomide and PS1145; or lenalidomide, PS1145and an RTK inhibitor are administered to patients that have becomeresistant to a cancer drug, e.g., drugs like erotinib or lapatinib, toproduce a strong antitumor effect.

In alternative embodiments, any NF-kB inhibitor can be used to practicethis invention, e.g., an antioxidant can be used to inhibit activationof NF-kB, e.g., including the compositions listed in Table 1:

TABLE 1 Antioxidants that have been shown to inhibit activation of NF-kBMolecule Reference a-Lipoic acid Sen et al, 1998; Suzukiet al, 1992a-tocopherol Islam et al, 1998 Aged garlic extract (allicin) Ide & Lau,2001; Langet al, 2004; Hasan et al, 20072-Amino-1-methyl-6-phenylimidazo[4,5- Yun et al, 2005 b]pyridine (PhIP)N-acetyldopamine dimers (from P. cicadae) Xu et al, 2006 AllopurinolGomez-Cabrera et al, 2006 Anetholdithiolthione Sen et al, 1996 ApocyninBarbieri et al, 2004 Apple juice/extracts Shi & Jiang, 2002; Daviset al,2006; Jung et al, 2009 Aretemsia p7F (5,6,3′,5′-tetramethoxy 7,4′- Leeet al, 2004 hydroxyflavone) Astaxanthin Lee et al, 2003 Autumn oliveextracts; olive leaf extracts Wang et al, 2007; Wanget al, 2008Avenanthramides (from oats) Guo et al, 2007; Sur et al, 2008 Bamboo culmextract Lee et al, 2008 Benidipine Matsubara & Hazegawa, 2004bis-eugenol Murakami et al, 2003 Bruguiera gymnorrhiza compounds Homhualet al, 2006 Butylated hydroxyanisole (BHA) Israël et al, 1992;Schulze-Osthoffet al, 1993 Cepharanthine Okamoto et al, 1994; Tamataniet al, 2007 Caffeic Acid Phenethyl Ester (3,4- Natarajan et al, 1996;Nagasaka et al, dihydroxycinnamic acid, CAPE) 2007 Carnosol Lo et al,2002; Huang et al, 2005 beta-Carotene Bai et al, 2005; Guruvayoorappan&Kuttan, 2007 Carvedilol Yang et al, 2003 Catechol Derivatives Suzuki &Packer, 1994; Zheng et al, 2008 Centaurea L (Asteraceae) extractsKaramenderes et al, 2007 Chalcone Liu et al, 2007 Chlorogenic acid Fenget al, 2005 5-chloroacetyl-2-amnio-1,3-selenazoles Nam et al, 2008Cholestin Lin et al, 2007 Chroman-2-carboxylic acid N-substituted Kwaket al, 2008 phenylamides Cocoa polyphenols Lee et al, 2006 Coffeeextract (3-methyl-1,2- Chung et al, 2007 cyclopentanedione) Crataeguspinnatifida polyphenols Kao et al, 2007 Curcumin (Diferulolylmethane);Singh & Aggarwal, 1995; Pae et al, dimethoxycurcumin; EF24 analog 2008;Kasinskiet al, 2008 Dehydroepiandrosterone (DHEA) Iwasaki et al, 2004;Liuet al, 2005 and DHEA-sulfate (DHEAS) Dibenzylbutyrolactone lignansCho et al, 2002 Diethyldithiocarbamate (DDC) Schreck et al, 1992Diferoxamine Sappey et al, 1995; Schreck et al, 1992 Dihydroisoeugenol;isoeugenol; Murakami et al, 1995; Park et al,epoxypseudoisoeugenol-2-methyl butyrate 2007; Ma et al, 2008Dihydrolipoic Acid Suzuki et al, 1992, 1995 Dilazep + fenofibric acidSonoki et al, 2003; Yanget al, 2005 Dimethyldithiocarbamates (DMDTC)Pyatt et al, 1998 Dimethylsulfoxide (DMSO) Kelly et al, 1994 DisulfiramSchreck et al, 1992 Ebselen Schreck et al, 1992 Edaravone Kokura et al,2005; Ariiet al, 2007; Yoshida et al, 2007 EPC-K1 (phosphodiestercompound of vitamin Hirano et al, 1998 E and vitamin C)Epigallocatechin-3-gallate (EGCG; green tea Lin & Lin, 1997; Yangetpolyphenols) al,1998; Hou et al, 2007 Ergothioneine Rahman et al, 2003Ethyl Pyruvate (Glutathione depletion) Song et al, 2004; Tsunget al,2005; Jimenez-Lopezet al, 2008 Ethylene Glycol Tetraacetic Acid (EGTA)Janssen et al, 1999 Eupatilin Lee et al, 2008 Exercise Goto et al, 2007Fisetin Park et al, 2006; Sunget al, 2007 Flavonoids (Crataegus;Boerhaavia diffusa Zhang et al, 2004; Chenet al, root; xanthohumol;Eupatorium arnottianum; 2004; Pandey et al, 2005; Albini et al,genistein; kaempferol; quercetin, daidzein; 2005; Colgate et al, 2006;Clavin et al, flavone; isorhamnetin; naringenin; 2007; Hamalainen et al,pelargonidin; finestin; Sophora flavescens; 2008; Zheng et al, 2008;Junget al, Seabuckthorn fruit berry) 2008; Mishra et al, 2008 Folic acidAu-Yeung et al, 2006 Gamma-glutamylcysteine synthetase (gamma- Manna etal, 1999 GCS) Ganoderma lucidum polysaccharides Zhang et al, 2003; Ho etal, 2007 Garcinol (from extract of Garcinia indica fruit Liao et al,2004 rind) Ginkgo biloba extract Chen et al, 2003 Glutathione Cho et al,1998; Schrecket al, 1992; Wang et al, 2007 Guaiacol (2-methoxyphenol)Murakami et al, 2007 Hematein Choi et al, 2003 Hinokitiol Byeon et al,2008 HMCO5 herbal extract Kim et al, 2007 Hydroquinone Pyatt et al,1998; Yanget al, 2006 23-hydroxyursolic acid Shin et al, 2004 IRFI 042(Vitamin E-like compound) Altavilla et al, 2001 Iron tetrakis Kang etal, 2001 Isosteviol Xu et al, 2008 Isovitexin Lin et al, 2005Isoliquiritigenin Kumar et al, 2007; Kimet al, 2008; Kim et al, 2008Justicia gendarussa root extract Kumar et al, 2011 Kallistatin Shen etal, 2008 Kangen-karyu extract Satoh et al, 2005; Yokozawa et al, 2007L-cysteine Mihm et al, 1991 Lacidipine Cominacini et al, 1997 LazaroidsMarubayashi et al, 2002 Ligonberries Wang et al, 2005 Lupeol Saleem etal, 2004; Leeet al, 2007 Lutein Kim et al, 2008 Magnolol Chen et al,2002; Ou et al, 2006; Kim et al, 2007 Maltol Yang et al, 2006 Manganesesuperoxide dismutase (Mn-SOD) Manna et al, 1998 Extract of the stem barkof Mangifera indica L. Leiro et al, 2004; Garridoet al, 2005 MelatoninGilad et al, 1998; Mohanet al, 1995; Li et al, 2005 21 (alpha,beta)-methylmelianodiol Zhou et al, 2007 Mulberry anthocyanins Chen etal, 2006 N-acetyl-L-cysteine (NAC) Schreck et al, 1991 Nacyselyn (NAL)Antonicelli et al, 2002 Nordihydroguaiaritic acid (NDGA) Brennan &O'Neill, 1998; Israël et al,1992; Schulze-Osthoff et al, 1993; Staaletal, 1993 Ochnaflavone Suh et al, 2006 Onion extract(2,3-dihydro-3,5-dihydroxy-6- Ban et al, 2007; Tang et al, 2008methyl-4H-pyranone) Orthophenanthroline Schreck et al, 1992N-(3-oxo-dodecanoyl) homoserine lactone Kravchenko et al, 2008Paricalcitol Tan et al, 2008 Phenolic antioxidants (Hydroquinone andtert- Ma et al, 2003 butyl hydroquinone) alkenylphenols from Piperobliquum Valdivia et al, 2008 alpha-phenyl-n-tert-butyl-nitrone (PEN)Kotake et al, 1998; Linet al, 2006 Phenylarsine oxide (PAO, tyrosinephosphatase Arbault et al, 1998 inhibitor) Phyllanthus urinariaChularojmontri et al, 2005; Shen et al, 2007 Phytosteryl ferulates (ricebran) Islam et al, 2008; Junget al, 2008 Piper longum Linn, extractSingh et al, 2007 Pitavastatin Tounai et al, 2007; Wang& Kitajima, 2007Prodelphinidin B2 3, 3′ di-O-gallate Hou et al, 2007 PterostilbeneCichocki et al, 2008; Panet al, 2009 Pyrrolinedithiocarbamate (PDTC)Schreck et al, 1992 Quercetin Musonda & Chipman, 1998; Shih et al, 2004;Garcia-Mediavillaet al, 2006; Ruiz et al, 2007; Min et al, 2007; Kim etal, 2007 Red orange extract Cimini et al, 2008 Red wine Blanco-Colio etal, 2000; Cui & He, 2004 Ref-1 (redox factor 1) Ozaki et al, 2002 Rg(3),a ginseng derivative Keum et al, 2003 Rotenone Schulze-Osthoff et al,1993 Roxithromycin Ueno et al, 2005; Ou et al, 2008 Rutin Kyung et al,2008 S-allyl-cysteine (SAC, garlic compound) Geng et al, 1997Salogaviolide (Centaurea ainetensis) Ghantous et al, 2008 Sauchinone Leeet al, 2003; Hwang et al, 2003 Schisandrin B Giridharan et all, 2011Silybin Gazak et al, 2007 Spironolactone Han et al, 2006 Strawberryextracts Wang et al, 2005 Taxifolin Wang et al, 2005 Tempol Cuzzocrea etal, 2004 Tepoxaline (5-(4-chlorophenyl)-N-hydroxy-(4- Kazmi et al, 1995;Ritchieet al, 1995 methoxyphenyl) -N-methyl-1H-pyrazole-3- propanamide)Thio avarol derivatives Amigo et al, 2007; Amigoet al, 2008 ThymoquinoneEl Gazzar et al, 2007; lSethi et al, 2008 Tocotrienol (palm oil) Wu etal, 2008 Tomato peel polysaccharide De Stefano et al, 2007 UDNglycoprotein (Ulmus davidiana Nakai) Lee & Lim, 2007 Vaccinium stamineum(deerberry) extract Wang et al, 2007 Vanillin(2-hydroxy-3-methoxybenzaldehyde) Murakami et al, 2007 Vitamin C Staalet al, 1993; Son et al, 2004 Vitamin B 6 Yanaka et al, 2005 Vitamin Eand derivatives Suzuki & Packer, 1993; Ekstrand- Hammarstrom et al,2007; Glauert, 2007 a-torphryl succinate Staal et al, 1993; Suzuki &Packer, 1993 a-torphryl acetate Suzuki & Packer, 1993 PMC(2,2,5,7,8-pentamethyl-6- Suzuki & Packer, 1993 hydroxychromane)Yakuchinone A and B Chun et al, 2002

In alternative embodiments, any proteasome inhibitor and/or proteaseinhibitor can be used to practice the invention, e.g., any proteasomeinhibitor and/or protease inhibitor that can inhibit Rel and/or NF-kBcan be used to practice this invention, e.g., including the compositionslisted in Table 2:

TABLE 2 Proteasome and proteases inhibitors that inhibit Rel/NF-kBMolecule References Proteasome inhibitors Peptide Aldehydes: Palombellaet al, 1994; Grisham et al, 1999; Jobin et al, 1998 ALLnL(N-acetyl-leucinyl-leucynil- norleucynal, MG101) LLM(N-acetyl-leucinyl-leucynil- methional) Z-LLnV(carbobenzoxyl-leucinyl-leucynil- norvalinal, MG115) Z-LLL(carbobenzoxyl-leucinyl-leucynil- leucynal, MG132) Lactacystine,beta-lactone Fenteany & Schreiber, 1998; Grisham et al, 1999 BoronicAcid Peptide Grisham et al, 1999; Iqbal et al, 1995 Dithiocarbamatecomplexes with Cvek & Dvorak, 2007 metals CEP-18770 Piva et al, 2007Ubiquitin Ligase Inhibitors Yaron et al, 1997 PS-341 (Bortezomib) Adams,2004 Salinosporamide A (1, NPI-0052) Macherla et al, 2005; Ahn et al,2007 Cyclosporin A Frantz et al, 1994; Kunz et al, 1995; Marienfeld etal, 1997; McCaffrey et al, 1994; Meyer et al, 1997; Wechsler et al, 1994FK506 (Tacrolimus) Okamoto et al, 1994; Venkataramen et al, 1995Deoxyspergualin Tepper et al, 1995 Disulfiram Lovborg et al, 2005 PT-110Momose et al, 2007 Protease inhibitors APNE(N-acetyl-DL-phenylalanine-b- Higuchi et al, 1995 naphthylester) BTEE(N-benzoyl L-tyrosine- Rossi et al, 1998 ethylester) DCIC(3,4-dichloroisocoumarin) D'Acquisto et al, 1998 DFP (diisopropylfluorophosphate) TPCK (N-a-tosyl-L-phenylalanine chloromethyl ketone)TLCK (N-a-tosyl-L-lysine chloromethyl ketone)

In alternative embodiments, any IκBα (nuclear factor of kappa lightpolypeptide gene enhancer in B-cells inhibitor, alpha) phosphorylationand/or degradation inhibitor can be used to practice this invention,e.g., including the compositions listed in Table 3:

TABLE 3 IκBα phosphorylation and/or degradation inhibitors MoleculePoint of Inhibition References Desloratadine; diphenhydramine HistamineH1 receptor Wu et al, 2004; Scadding, 2005; Roumestan et al, 2008Bikunin LPS receptor agonists Kobayashi, 2006; Kanayama et al, 2007 RonTyrosine kinase receptor Suppresses TNF Lentsch et al, 2007 productionTAK-242 TLR4 intracellular Kawamoto et al, 2008 domain Salmeterol,fluticasone propionate beta2 agonists Baouz et al, 2005 CPU0213Endothelin receptor He et al, 2006 antagonist Doxazosinalpha1-adrenergic Hui et al, 2007 receptor antagonist Erbinoverexpression NOD2 inhibitor McDonald et al, 2005 Protein-boundpolysaccharide LPS-CD14 interaction Asai et al, 2005 from basidiomycetesAnti-CD 146 antibody AA98 upstream of IKK Bu et al, 2006 Calagualine(fern derivative) upstream of IKK Manna et al, 2003 (TRAF2-NIK) NS3/4A(HCV protease) upstream of IKK Karayiannis, 2005 golli BG21 (product ofmyelin upstream of IKK (PKC) Feng et al, 2004 basic protein) NPM-ALKoncoprotein Traf2 inhibition Horie et al, 2004 NS5A (Hepatitis C virus)Traf2 inhibition Park et al, 2002 LY29 and LY30 PI3 Kinase inhibitorsChoi et al, 2004 Shiga toxin (Enterohemorrhagic E PI3 Kinase inhibitorGobert et al, 2007 coli) Evodiamine (Evodiae Fructus AKT-IKK interactionTakada et al, 2005 component) Rituximab (anti-CD20 antibody)up-regulates Raf-1 Jazirehi et al, 2005 kinase inhibitor Kinasesuppressor of ras (KSR2) MEKK3 inhibitor Channavajhala et al, 2005Cholecystokinin ocatpeptide p38 kinase Li et al, 2007 (CCK-8) M2L(Vaccinia virus) ERK2 inhibitor Gedey et al, 2006; Hinthong et al, 2008Pefabloc (serine protease inhibitor) upstream of IKK Tando et al, 2002Rocaglamides (Aglaia derivatives) upstream of IKK Baumann et al, 2002Ymer Binds to Ub-RIP Bohgaki et al, 2007 Epoxyquinol B TAK1 crosslinkerKamiyama et al, 2008 Betaine NIK/IKK Go et al, 2004, 2007 TNAP NIK Hu etal, 2005 Selected peptides NEMO binding to Ub Wyler et al, 2007Desflurane IKK complex formation Li et al, 2008 with TNF-R1 GeldanamycinIKK complex formation Chen et al, 2002 Grape seed proanthocyanidins IKKaactivity Mantena & Katiyar, 2006; Sharma et al, 2007; Cheng et al, 2007;Xu et al, 2008 Laretia acaulis azorellane IKKa activity Borquez et al,2007 diterpenoids MC160 (Molluscum contagiosum IKKa activity Nichols &Shisler, 2006 virus) NS5B (Hepatitis C protein) IKKa activity Choi etal, 2006 Pomegranate fruit extract IKKa activity Afaq et al, 2004; Khanet al, 2006 Tetrandine (plant alkaloid) IKKa activity Ho et al, 2004;Xueet al, 2008; Lin et al, 2008 BMS-345541 (4(2′- IKKa and IKKb kinaseBurke et al, 2002; Yang et Aminoethyl)amino-1,8- activity al, 2006;Beaulieu et al, dimethylimidazo(1,2-a) 2006 quinoxaline) and 4-aminoderivatives 1-O-acetylbritannilactone IKKb activity Liu et al, 20072-amino-3-cyano-4-aryl-6-(2- IKKb activity Murata et al,hydroxy-phenyl)pyridine 2003, 2004, 2004 derivatives Acrolein IKKbactivity/p50 DNA Vallacchi et al, binding 2005; Lambert et al, 2007Anandamide IKKb activity Sancho et al, 2003 AS602868 IKKb activityFrelin et al, 2003: Griessinger et al, 2007 Cobrotoxin IKKb activity/p50DNA Park et al, 2005 binding Core protein (Hepatitis C) IKKb activityJoo et al, 2005; Shrivastava et al, 1998 1-[2-cyano-3,12-dioxooleana-IKKb activity Yore et al, 2006 1,9(11)-dien-28-oyl] imidazoleDihydroxyphenylethanol IKKb activity Guichard et al, 2006 Herbimycin AIKKb activity Iwasaki et al, 1992; Mahon & O'Neill, 1995; Ogino et al,2004 Inhibitor 22 IKKb activity Baxter et al, 2004 Isorhapontigenin IKKbactivity Li et al, 2005 Manumycin A IKKb activity Bernier et al, 2005;Frassanito et al, 2005 6-methyl-2-propolyimino-6,7- IKKb Kim et al, 2008dihydro-5H- benzo[1,3]oxathiol-4-one MLB120 (small molecule) IKKbactivity Nagashima et al, 2006 Naphthopyrones (6- IKKb activity Fulmeret al, 2008 methoxycomaparvin and 6- methooxycomaparvin 5-methyl ether)Novel Inhibitor IKKb activity Kamon et al, 2004 vIRF3 (KSHV) IKKbactivity Seo et al, 2004 Nitric oxide IKKb activity/IkB Katsuyama et al,phosphorylation 1998; Matthews et al, 1996; Spieker & Liao, 1999;Reynaert et al, 2004 SC-514 (small molecule) IKKb activity Kishore etal, 2003 Thienopyridine IKKb activity Morwick et al, 2006Acetyl-boswellic acids IKK activity Syrovets et al, 2004, 2005Amino-pyrimidine derivative IKK activity Karin et al, 2004Benzoimidazole derivative IKK activity Karin et al, 2004 BMS-345541 IKKactivity Burke et al, 2003 Butein IKKb activity Pandey et al, 2007Beta-carboline IKK activity Yoon et al, 2005 CYL-19s and CYL-26z, twoIKK activity Huang et al, 2004 synthetic alpha-methylene-gamma-butyrolactone derivatives ACHP (2-amino-6-[2- IKKb activity (ATPSanda et al, 2006 (cyclopropylmethoxy)-6- analog)hydroxyphenyl]-4-piperidin-4-yl nicotinonitrile Berberine IKKb activityHu et al, 2007; Yi et al, 2008; Pandey et al, 2008 Compound A IKKbactivity (ATP Ziegelbauer et al, 2005 analog) Flavopiridol IKK activityand RelA Takada & Aggarwal, phosphor. 2003 Cyclopentones IKKb activityBickley et al, 2004 Dehydroascorbic acid (Vitamin C) IKKb activityCarcamo et al, 2004 Gossypyin or Gossypium extracts IKKb activityKunnumakkara et al, 2007; Ji et al, 2008 M protein (SARS-CornonavirusIKKb activity Fang et al, 2007 protein) IMD-0354 IKKb activity Tanaka etal, 2004, 2006; Inayama et al, 2006 Jesterone dimer IKKb activity; DNALiang et al, 2003, 2006 binding KINK-1 IKKb activity Schon et al, 2008LCY-2-CHO IKKb activity Ho et al, 2007 Prolyl hydroxylase-1 IKKbactivity Cummins et al, 2006 Naphthopyrones (Echinoderm IKKb activityFolmer et al, 2007 Comanthus parvicirrus) Neuropeptides CGRP, PACAP andIKKb activity Ding et al, 2007 VIP PS-1145 (MLN1145) IKKb activityHideshima et al, 2002 2-[(aminocarbonyl)amino]-5-(4- IKKb activityBonafoux et al, fluorophenyl)-3- 2005; Podolin et al, 2005thiophenecarboxamides (TPCA-1) 1′-Acetoxychavicol acetate IKK activityIchikawa et al, (Languas galanga) 2005; Ito et al, 200517-Acetoxyjolkinolide B IKK activity Yan et al, 2008 Acute alcoholexposure IKK activity Mandrekar et al, 2007 Anacardic acid (6-nonadecyl-IKK activity Sung et al, 2008 salicylic acid) Apigenin (plant flavinoid)IKK activity Shukla & Gupta, 2004; Yoon et al, 2006 Asiatic acid IKKactivity Yun et al, 2008 Cardamomin IKK activity Lee et al, 2005 CDDO-Me(synthetic triterpenoid) IKK activity Shishodia et al, 2006 CHS 828(anticancer drug) IKK activity Olsen et al, 2004 CML-1 IKK activity Moet al, 2006 Compound 5 (Uredio- IKK activity Roshak et al, 2002thiophenecarboxamide derivative) CT20126 IKK activity/NIK Lee et al,2008 Diaylpyridine derivative IKK activity Murata et al, 20033,4-dihydroxybenzalacetone (from IKK activity Sung et al, 2008 Chaga)Diosgenin IKK activity Shishodia & Aggarwal, 2005; Liagre et al, 2005E3-14.7K (Adenovirus) IKK activity Li et al, 1999 E3-10.4K/14.5K(Adenovirus) IKK activity Friedman & Horwitz, 2002 E7 (humanpapillomavirus) IKK activity Spitkovsky et al, 2002 FuronaphthoquinoneIKK activity Shin et al, 2006 3-Formylchromone IKKb activity/p65 DNAYadav et al, 2011 binding Guggulsterone IKK activity Ichikawa &Aggarwal, 2006; Deng, 2007; Lv et al, 2008; Lee et al, 2008 HB-EGF(Heparin-binding IKK activity Mehta & Besner, 2003 epidermal growthfactor-like growth factor) Falcarindol IKK activity Shiao et al, 2005Hammerhead ribozyme to IKKa/b IKK activity Yang et al, 2007 Hepatocytegrowth factor IKK activity Min et al, 2005; Gong et al, 2006 HonokiolIKK activity Tse et al, 2005; Munroe et al, 2007 Humulone IKK activityLee et al, 2007 Hypoestoxide IKK activity Ojo-Amaize et al, 2001Indolecarboxamide derivative IKK activity Karin et al, 2004 Labdanediterpenoids IKK activity Giron et al, 2008 LF15-0195 (analog of 15- IKKactivity Yang et al, 2003 deoxyspergualine) gamma-mangostin (fromGarcinia IKK activity Nakatani et al, 2004 mangostana) Garcinone B IKKactivity Yamakuni et al, 2005 (Amino)imidazolylcarboxaldehyde IKKactivity Karin et al, 2004 derivative Imidazolylquinoline- IKK activityKarin et al, 2004 carboxaldehyde derivative Kahweol IKK activity Kim etal, 2004 Kava (Piper methysticum) IKK activity Folmer et al, 2006derivatives Lead IKK activity Xu et al, 2006 Marasmius oreades liquidextract IKK activity Petrova et al, 2008 Menatetrenone (vitamin K2 IKKactivity Ozaki et al, 2007 analogue) Metformin IKK activity Huang et al,2008 Mild hypothermia IKK activity Han et al, 2003 ML120B IKK activityCatley et al, 2006 Morin (3,5,7,2′,4′- IKK activity Manna et al, 2007Pentahydroxyflavone) Morusin IKK activity Lee et al, 2008 MX781(retinoid antagonist) IKK activity Bayon et al, 2003 N-acetylcysteineIKK activity Oka et al, 2000 Nitrosylcobalamin (vitamin B12 IKK activityChawla-Sarkar et al, 2003 analog) NSAIDs IKK activity Takada et al, 2004Hepatits C virus NS5B IKK activity Choi et al, 2006 PAN1 (aka NALP2 orPYPAF2) IKK activity Bruey et al, 2004 Pectin (citrus) IKK activity Chenet al, 2006 Pinitol IKK activity Sethi et al, 2008 PMX464 IKK activityCallister et al, 2008 Pyrazolo[4,3-c]quinoline IKK activity Karin et al,2004 derivative Pyridooxazinone derivative IKK activity Karin et al,2004 N-(4-hydroxyphenyl) retinamide IKK activity Shishodia et al, 2005;Kuefer et al, 2007 Scytonemin IKK activity Stevenson et al, 2002Semecarpus anacardiu extract IKK activity Singh et al, 2006 SPC-839 IKKactivity Palanki et al, 2002 Sulforaphane and IKK activity Xu et al,phenylisothiocyanate 2005; Murakami et al, 2007; Liu et al, 2008: Hayeset al, 2008 Survanta (Surfactant product) IKK activity Raychaudhuri etal, 2003 Torque Teno virus ORF2 IKK activity Zheng et al, 2007Piceatannol IKK activity Islam et al, 2004 Plumbagin(5-hydroxy-2-methyl- IKK activity Sandur et al, 2006 1,4-naphthoquinone)IKKb peptide to NEMO binding IKK-NEMO interaction May et al, 2000 domainNEMO CC2-LZ peptide NEMO oligomerization Agou et al, 2004 AGRO100(G-quadraplex NEMO binding Girvan et al, 2006 oligodeoxynucleotide) PTEN(tumor suppressor) Activation of IKK Gustin et al, 2001 Theaflavin(black tea component) Activation of IKK Aneja et al, 2004; Ukil et al,2006; Kalra et al, 2007 Tilianin Activation of IKK Nam et al, 2005Withanolides Activation of IKK Ichikawa et al, 2006 Zerumbone Activationof IKK Takada et al, 2005 Silibinin IKKa activity; nuclear Dhanalakshmiet al, translocation 2002; Singh et al, 2004; Min et al, 2007Sulfasalazine IKKa and IKKb kinase Wahl et al, activity 1998: Weber etal, 2000 Sulfasalazine analogs IKK kinase activity Habens et al, 2005Quercetin IKK activity Peet & Li, 1999 Rosmarinic acid IKK activity Leeet al, 2006 Staurosporine IKK activity Peet & Li, 1999 gamma-TocotrienolIKK activity Shah & Sylvester, 2005; Ahn et al, 2006 Wedelolactone IKKactivity Kobori et al, 2003 Betulinic acid IKKa activity and p65 Takada& Aggarwal, phosphorylation 2003; Rabi et al, 2008 Ursolic acid IKKaactivity and p65 Shishodia et al, phosphorylation 2003; Manu & Kuttan,2008 Thalidomide (and thalidomide IKK activity Keifer et al, 2001; Ge etanalogs) al, 2006; Carcache de- Blanco et al, 2007 Salubrinal IKK Huanget al, 2011 activity/degradation Fas-associated factor-1 IKK assemblyPark et al, 2007 Interleukin-10 Reduced IKKa and Tabary et al, 2003 IKKbexpression MC160 (molluscum contagiosum Reduced IKKa Nichols & Shisler,2006 virus) expression Monochloramine and glycine Oxidizes IkB Kim etal, chloramine (NH2Cl) 2005; Midwinter et al, 2006 GS143 Blocks IkBNakajima et al, ubiquitylation 2008; Hirose et al, 2008 SalmonellaSecreted Factor L Blocks IkB Le Negrate et al, 2008 ubiquitylationAnethole Phosphorylation Chainy et al, 2000 Anti-thrombin IIIPhosphorylation Oelschlager et al, 2002 Artemisia vestitaPhosphorylation Sun et al, 2006 Aspirin, sodium salicylatePhosphorylation, Frantz & O'Neill, IKKbeta 1995; Kopp & Ghosh, 1994; Yinet al, 1998 Azidothymidine (AZT) Phosphorylation Ghosh et al, 2003;Kurokawa et al, 2005 Baoganning Phosphorylation Tan et al, 2005BAY-11-7082 Phosphorylation Pierce et al, 1997(E3((4-methylphenyl)-sulfonyl)-2- propenenitrile) BAY-117083Phosphorylation Pierce et al, 1997 (E3((4-t-butylphenyl)-sulfonyl)-2-propenenitrile) Benzyl isothiocyanate Phosphorylation Srivastava &Singh, 2004 Black raspberry extracts (cyanidin Phosphorylation Huang etal, 3-O-glucoside, cyanidin 3-O- 2002; Hecht et al, 2006(2(G)-xylosylrutinoside), cyanidin 3-O-rutinoside) Buddlejasaponin IVPhosphorylation Won et al, 2006 Cacospongionolide B PhosphorylationPosadas et al, 2003 Calagualine Phosphorylation Manna et al, 2003 Carbonmonoxide Phosphorylation Sarady et al, 2002 Carboplatin PhosphorylationSingh & Bhat, 2004 Cardamonin Phosphorylation Israf et al, 2006Chorionic gonadotropin Phosphorylation Manna et al, 2000 CordycepinPhosphorylation Kim et al, 2006; Huang et al., 2007 Crassocephalumrabens Phosphorylation Hou et al., 2007 galactolipid Cycloepoxydon;1-hydroxy-2- Phosphorylation Gehrt et al, 1998 hydroxymethyl-3-pent-1-enylbenzene Cytomegalovirus Phosphorylation Jarvis et al, 2006 DecursinPhosphorylation Kim et al, 2006 Delphinidin Phosphorylation Syed et al,2008 Dexanabinol Phosphorylation Juttler et al, 2004 DigitoxinPhosphorylation Srivastava et al, 2004; Jagielska et al, 2009Dihydrotestosterone Phosphorylation Xu et al, 2011 Diterpenes(synthetic) Phosphorylation Chao et al, 2005 Docosahexaenoic acidPhosphorylation Chen et al, 2005; Zand et al, 2008 Entamoeba histolyticaPhosphorylation Kammanadiminti & Chadee, 2006 Extensively oxidized lowdensity Phosphorylation Brand et al, 1997; Page et lipoprotein (ox-LDL),4- al, 1999 Hydroxynonenal (HNE) FBD Phosphorylation Lin et al, 2008FHIT (Fragile histidine triad Phosphorylation Nakagawa & Akao, 2006protein) Fructus Ligustrum lucidi Phosphorylation An et al, 2007Gabexate mesilate Phosphorylation Uchiba et al, 2003 [6]-gingerol;casparol Phosphorylation Kim et al, 2005; Aktan et al, 2006; Ishiguro etal, 2007 Gleditsia sinensis thorns extract Phosphorylation Ha et al,2008 Gleevec (Imatanib) Phosphorylation Wolf et al, 2005 Glossogynetenuifolia Phosphorylation Wu et al, 2004; Haet al, 2006 GuggulsteronePhosphorylation Shishodia & Aggarwal, 2004 4-hydroxy-3,6,7,8,3′,4′-Phosphorylation Lai et al, 2007 hexamethoxyflavone HydroquinonePhosphorylation Kerzic et al, 2003 Ibuprofen Phosphorylation Palayoor etal, 1998 Indirubin-3′-oxime Phosphorylation Mak et al, 2004 Inonotusobliquus ethanol extract Phosphorylation Kim et al, 2007Interferon-alpha Phosphorylation Manna et al, 2000 Inhaled isobutylnitrite Phosphorylation Ponnappan et al, 2004 Kaempferol PhosphorylationGarcia-Mediavilla et al, 2006; Kim et al, 2007 Kushen flavonoids andkurarinone Phosphorylation Han et al, 2006 Licorce extractsPhosphorylation Kim et al, 2006: Kwon et al, 2007 MelatoninPhosphorylation Alonso et al, 2006; Tamura et al, 2009 Marine naturalproducts (several) IKKb/proteasome Folmer et al, 2009 MethotrexatePhosphorylation Majumdar & Aggarwal, 2001; Yozai et al, 2005Monochloramine Phosphorylation Omori et al, 2002 Nafamostat mesilatePhosphorylation Noguchi et al, 2003 Obovatol Phosphorylation Lee et al,2008 Oleandrin Phosphorylation Manna et al, 2000; Sreeivasan et al, 2003Oleanolic acid (Aralia elata) Phosphorylation Suh et al, 2007 Omega 3fatty acids Phosphorylation Novak et al, 2003 Panduratin A (fromKaempferia Phosphorylation Yun et al, 2003 pandurata, Zingiberaceae)Petrosaspongiolide M Phosphorylation Posadas et al, 2003 PinosylvinPhosphorylation Lee et al, 2006 Plagius flosculosus extractPhosphorylation Calzado et al, 2005 polyacetylene spiroketal Phytic acid(inositol Phosphorylation Ferry et al, 2002 hexakisphosphate)Pomegranate fruit extract Phosphorylation Ahmed et al, 2005Prostaglandin A1 Phosphorylation/IKK Rossi et al, 1997, 2000Protocatechuic Aldehyde Phosphorylation Xu et al, 201120(S)-Protopanaxatriol Phosphorylation Oh et al, 2004; Leeet al,(ginsenoside metabolite) 2005 Rengyolone Phosphorylation Kim et al, 2006Rottlerin Phosphorylation Kim et al, 2005; Torricelli et al, 2008Saikosaponin-d Phosphorylation; Leung et al, 2005; Dang et Increased IkBal, 2007 Saline (low Na+ istonic) Phosphorylation Tabary et al, 2003Salvia miltiorrhizae water-soluble Phosphorylation Kim et al, 2005extract Sanguinarine Phosphorylation Chaturvedi et al, 1997(pseudochelerythrine, 13-methyl- [1,3]-benzodioxolo-[5,6-c]-1,3-dioxolo-4,5 phenanthridinium) Scoparone Phosphorylation Jang et al, 2005Sesaminol glucosides Phosphorylation Lee et al, 2006 ShikoninsPhosphorylation Nam et al, 2008 Silymarin Phosphorylation Manna et al,1999; Saliou et al, 1998 Snake venom toxin (Vipera Phosphorylation Sonet al, 2007 lebetina turanica) SOCS1 Phosphorylation Kinjyo et al, 2002;Nakagawa et al, 2002 Spilanthol Phosphorylation Wu et al, 2008 Statins(several) Phosphorylation Hilgendorff et al, 2003; Han et al, 2004;Planavila et al, 2005 Sulindac IKK/Phosphorylation Yamamato et al, 1999THI 52 (1-naphthylethyl-6,7- Phosphorylation Kang et al, 2003dihydroxy-1,2,3,4- tetrahydroisoquinoline) 1,2,4-thiadiazolidinederivatives Phosphorylation Manna et al, 2004 Tomatidine PhosphorylationChiu & Lin, 2008 Vesnarinone Phosphorylation Manna & Aggarwal, 2000;Harada et al, 2005 Xanthoangelol D Phosphorylation Sugii et al, 2005YC-1 Phosphorylation Huang et al, 2005 YopJ (encoded by YersiniaDeubiquintinase for Schesser et al, pseudotuberculosis) IkBa;Acetylation of 1998; Zhou et al, IKKbeta 2005; Mittal et al, 2006;Mukherjee & Orth, 2008 Osmotic stress IkB ubiquitination Huangfu et al,2007 Acetaminophen Degradation Mancini et al, 2003 Activated Protein C(APC) Degradation Yuksel et al, 2002 Alachlor DegradationShimomura-Shimizu et al, 2005 Allylpyrocatechol Degradation Sarkar etal, 2008 a-melanocyte-stimulating hormone Degradation Manna & Aggarwal,1998 (a-MSH) Amentoflavone Degradation Banerjee et al, 2002;Guruvayoorappan & Kuttan, 2007 Angelica dahurica radix extractDegradation Kang et al, 2006 Apple extracts Degradation/proteasome Yoon& Liu, 2007 Artemisia capillaris Thunb extract Degradation Hong et al,2004; Kim et (capillarisin) al, 2007; Leeet al, 2007 Artemisia iwayomogiextract Degradation Kim et al, 2005 L-ascorbic acid Degradation Han etal, 2004 Antrodia camphorata Degradation Hseu et al, 2005 AucubinDegradation Jeong et al, 2002 Baicalein Degradation Ma et al, 2004N-(quinolin-8- Degradation Xie et al, 2007 yl)benzenesulfonamindesbeta-lapachone Degradation Manna et al, 1999 Blackberry extractDegradation Pergola et al, 2006 1-Bromopropane Degradation Yoshida etal, 2006 Buchang-tang Degradation Shin et al, 2005 Capsaicin(8-methyl-N-vanillyl-6- Degradation Singh et al, 1996; Mori etnonenamide) al, 2006; Kang et al, 2007 Catalposide Degradation Kim etal, 2004 Clerodendron trichotomum Degradation Park & Kim, 2007 TunbergLeaves Clomipramine/imipramine Degradation Hwang et al, 2008 Coptidisrhizoma extract Degradation Kim et al, 2007 Cyclolinteinone (spongeDegradation D'Acquisto et al, 2000 sesterterpene) DA-9601 (Artemisiaasiatica Degradation Choi et al, 2006 extract) Diamide (tyrosinephosphatase Degradation Toledano & Leonard, inhibitor) 1991; Singh &Aggarwal, 1995 Dihydroarteanniun Degradation Li et al, 2006 DobutamineDegradation Loop et al, 2004 Docosahexaenoic acid Degradation Weldon etal, 2006 E-73 (cycloheximide analog) Degradation Sugimoto et al, 2000Ecabet sodium Degradation Kim et al, 2003 Electrical stimulation ofvagus Degradation Guarini et al, 2003 nerve Emodin (3-methyl-1,6,8-Degradation Kumar et al, trihydroxyanthraquinone) 1998; Huang et al,2004 Ephedrae herba (Mao) Degradation Aoki et al, 2005 Equol DegradationKang et al, 2005 Erbstatin (tyrosine kinase Degradation Natarajan et al,1998 inhibitor) Estrogen (E2) Degradation/and various Sun et al, othersteps 1998; Kalaitzidis & Gilmore, 2005; Steffan et al, 2006 Ethacrynicacid Degradation (and DNA Han et al, 2004 binding) FludarabineDegradation Nishioka et al, 2007 Fosfomycin Degradation Yoneshima et al,2003 Fungal gliotoxin Degradation Pahletal, 1999 Gabexate mesilateDegradation Yuksel et al, 2003 Gamisanghyulyunbueum Degradation Shin etal, 2005 Genistein (tyrosine kinase Degradation; caspase Natarajan etal, inhibitor) cleavage of IkBa 1998; Baxa & Yoshimura, 2003 GenipinDegradation Koo et al, 2004 Glabridin Degradation Kang et al, 2004Ginsenoside Re Degradation Zhang et al, 2007 Glimepiride DegradationSchiekofer et al, 2003 Glucosamine (sulfate or Degradation Largo et al,2003; Rafi et carboxybutyrylated) al, 2007; Rajapakse et al, 2008gamma-glutamylcysteine Degradation Manna et al, 1999 synthetaseGlutamine Degradation Singleton et al, 2005; Fillmann et al, 2007; Chenet al, 2008 Glycochenodeoxycholate Degradation Bucher et al, 2006 Guaveleaf extract Degradation Choi et al, 2008 Gumiganghwaltang DegradationKim et al, 2005 Gum mastic Degradation He et al, 2007 Heat shockprotein-70 Degradation Chan et al, 2004; Shi et al, 2006 Herbal mixture(Cinnamomi Degradation Jeong et al, 2008 ramulus, Anemarrheriae rhizoma,Officinari rhizoma) Hypochlorite Degradation Mohri et al, 2002 IbudilastDegradation Kiebala & Maggirwar, 1998 IL-13 Degradation Manna &Aggarwal, 1998 Incensole acetate Degradation Moussaieff et al, 2007Intravenous immunoglobulin Degradation Ichiyama et al, 2004Isomallotochromanol and Degradation Ishii et al, 2003 isomallotochromeneK1L (Vaccinia virus protein) Degradation Shisler & Jin, 2004 Kochiascoparia fruit (methanol Degradation Shin et al, 2004 extract)Kummerowia striata (Thunb.) Degradation Tao et al, 2008 Schindl (ethanolextract) Leflunomide metabolite (A77 Degradation Manna & Aggarwal, 19991726) Lidocaine Degradation Feng et al, 2007; Lahat et al, 2008 LipoxinA4 Degradation Zhang et al, 2007 Losartan Degradation/NF-kB Chen et al,2002; Zhu et expression al, 2007 Low level laser therapy DegradationRizzi et al, 2006 LY294002 (PI3-kinase Degradation Park et al, 2002inhibitor) [2-(4-morpholinyl)-8- phenylchromone] MC159 (Molluscumcontagiosum Degradation of IkBb Murao & Shisler, 2005 virus) MelatoninDegradation Zhang et al, 2004 Meloxicam Degradation Liu et al, 20075′-methylthioadenosine Degradation Hevia et al, 2004 MidazolamDegradation Kim et al, 2006 Momordin I Degradation Hwang et al, 2005Morinda officinalis extract Degradation Kim et al, 2005 Mosla diantheraextract Degradation Lee et al, 2006 Mume fructus extract DegradationChoi et al, 2007 Murr1 gene product Degradation Ganesh et al, 2003Neurofibromatosis-2 (NF-2; Degradation Kim et al, 2002 merlin) proteinOpuntia ficus indica va saboten Degradation Lee et al, 2006 extractOzone (aqueous) Degradation Huth et al, 2007 Paeony total glucosidesDegradation Chen et al, 2007 Pectenotoxin-2 Degradation Kim et al, 2008Penetratin Degradation Letoya et al, 2006 Pervanadate (tyrosinephosphatase Degradation Singh & Aggarwal, inhibitor) 1995; Singh et al,1996 Phenylarsine oxide (PAO, tyrosine Degradation Mahboubi et al,phosphatase inhibitor) 1998; Singh & Aggarwal, 1995 beta-Phenylethyl(PEITC) and 8- Degradation Rose et al, 2005 methylsulphinyloctylisothiocyanates (MSO) (watercress) Phenytoin Degradation Kato et al,2005 c-phycocyanin Degradation Cherng et al, 2007 Platycodin saponinsDegradation Ahn et al, 2005; Leeet al, 2008 Polymeric formulaDegradation de Jong et al, 2007 Polymyxin B Degradation Jiang et al,2006 Poncirus trifoliata fruit extract Degradation; Shin et al, 2006;Kim et phosphorylation of IkBa al, 2007 Probiotics Degradation Petrof etal, 2004 Pituitary adenylate cyclase- Degradation Delgado & Ganea, 2001activating polypeptide (PACAP) Prostaglandin 15-deoxy- DegradationCuzzocrea et al, Delta(12,14)-PGJ(2) 2003; Chatterjee et al, 2004Prodigiosin (Hahella chejuensis) Degradation Huh et al, 2007 PS-341Degradation/proteasome Hideshima et al, 2002 Radix asari extractDegradation Song et al, 2007 Radix clematidis extract Degradation Lee etal, 2009 Resiniferatoxin Degradation Singh et al, 1996 SabaeksanDegradation Choi et al, 2005 SAIF (Saccharomyces boulardii DegradationSougioultzis et al, 2006 anti-inflammatory factor) Sanguis DraconisDegradation Choy et al, 2007 San-Huang-Xie-Xin-Tang Degradation Shih etal, 2007 Schisandra fructus extract Degradation Kang et al, 2006; Quo etal, 2008 Scutellarin Degradation Tan et al, 2007 Sesquiterpene lactonesDegradation Hehner et al, 1998; Whan (parthenolide; ergolide; Han et al,2001; Schorr et guaianolides; alpha-humulene; al, 2002; Medeiros et al,trans-caryophyllene) 2007 Sevoflurane/isoflurane Degradation Boost etal, 2009 Siegeskaurolic acid (from Degradation Park et al, 2007Siegesbeckia pubescens root) ST2 (IL-1-like receptor secretedDegradation Takezako et al, 2006 form) Synadenium carinatum latex lectinDegradation Rogerio et al, 2007 Taiwanofungus camphoratus DegradationLiu et al, 2007 Taurene bromamine Degradation Tokunaga et al, 2007Thiopental Degradation Loop et al, 2002 Tipifarnib Degradation Xue etal, 2005 Titanium Degradation Yang et al, 2003 TNP-470 (angiogenesisinhibitor) Degradation Mauriz et al, 2003 Stinging nettle (Urticadioica) Degradation Riehemann et al, 1999 plant extracts Trichomomasvaginalis infection Degradation Chang et al, 2004 Triglyceride-richlipoproteins Degradation Kumwenda et al, 2002 Tussilagone (Farfaraefios) Degradation Lim et al, 2008 U0126 (MEK inhibitor) DegradationTakaya et al, 2003 Ursodeoxycholic acid Degradation Joo et al, 2004Xanthium strumarium L. Degradation Kim et al, 2005; Yoon et (methanolextract) al, 2008 Yulda-Hanso-Tang Degradation Jeong et al, 2007 ZincDegradation Uzzo et al, 2006; Bao et al, 2006 Molluscum contagiosumvirus IkBbeta degradation Murao & Shisler, 2005 MC159 protein Vasoactiveintestinal peptide Degradation (and CBP- Delgado & Ganea, RelAinteraction) 2001; Delgado, 2002 HIV-1 Vpu protein TrCP ubiquitin ligaseBour et al, 2001 inhibitor Epoxyquinone A monomer IKKb/DNA binding Lianget al, 2006 Ro106-9920 (small molecule) IkBa ubiqutination Swinney etal, 2002 inhibitor Furonaphthoquinone IKK activity Shin et al, 2006

Pharmaceutical Compositions

In alternative embodiments, the invention provides pharmaceuticalcompositions for practicing the methods of the invention, e.g.,pharmaceutical compositions for overcoming or diminishing or preventingGrowth Factor Inhibitor (GFI) resistance in a cell, or, a method forincreasing the growth-inhibiting effectiveness of a Growth Factorinhibitor on a cell, or, a method for re-sensitizing a cell to a GrowthFactor Inhibitor.

In alternative embodiments, compositions used to practice the methods ofthe invention are formulated with a pharmaceutically acceptable carrier.In alternative embodiments, the pharmaceutical compositions used topractice the methods of the invention can be administered parenterally,topically, orally or by local administration, such as by aerosol ortransdermally. The pharmaceutical compositions can be formulated in anyway and can be administered in a variety of unit dosage forms dependingupon the condition or disease and the degree of illness, the generalmedical condition of each patient, the resulting preferred method ofadministration and the like. Details on techniques for formulation andadministration are well described in the scientific and patentliterature, see, e.g., the latest edition of Remington's PharmaceuticalSciences, Maack Publishing Co, Easton Pa. (“Remington's”).

Therapeutic agents used to practice the methods of the invention can beadministered alone or as a component of a pharmaceutical formulation(composition). The compounds may be formulated for administration in anyconvenient way for use in human or veterinary medicine. Wetting agents,emulsifiers and lubricants, such as sodium lauryl sulfate and magnesiumstearate, as well as coloring agents, release agents, coating agents,sweetening, flavoring and perfuming agents, preservatives andantioxidants can also be present in the compositions.

Formulations of the compositions used to practice the methods of theinvention include those suitable for oral/nasal, topical, parenteral,rectal, and/or intravaginal administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will vary depending upon the host being treated, theparticular mode of administration. The amount of active ingredient whichcan be combined with a carrier material to produce a single dosage formwill generally be that amount of the compound which produces atherapeutic effect.

Pharmaceutical formulations used to practice the methods of theinvention can be prepared according to any method known to the art forthe manufacture of pharmaceuticals. Such drugs can contain sweeteningagents, flavoring agents, coloring agents and preserving agents. Aformulation can be admixtured with nontoxic pharmaceutically acceptableexcipients which are suitable for manufacture. Formulations may compriseone or more diluents, emulsifiers, preservatives, buffers, excipients,etc. and may be provided in such forms as liquids, powders, emulsions,lyophilized powders, sprays, creams, lotions, controlled releaseformulations, tablets, pills, gels, on patches, in implants, etc.

Pharmaceutical formulations for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art inappropriate and suitable dosages. Such carriers enable thepharmaceuticals to be formulated in unit dosage forms as tablets,geltabs, pills, powder, dragees, capsules, liquids, lozenges, gels,syrups, slurries, suspensions, etc., suitable for ingestion by thepatient. Pharmaceutical preparations for oral use can be formulated as asolid excipient, optionally grinding a resulting mixture, and processingthe mixture of granules, after adding suitable additional compounds, ifdesired, to obtain tablets or dragee cores. Suitable solid excipientsare carbohydrate or protein fillers include, e.g., sugars, includinglactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,potato, or other plants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; andgums including arabic and tragacanth; and proteins, e.g., gelatin andcollagen. Disintegrating or solubilizing agents may be added, such asthe cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound (i.e., dosage). Pharmaceutical preparations used topractice the methods of the invention can also be used orally using,e.g., push-fit capsules made of gelatin, as well as soft, sealedcapsules made of gelatin and a coating such as glycerol or sorbitol.Push-fit capsules can contain active agents mixed with a filler orbinders such as lactose or starches, lubricants such as talc ormagnesium stearate, and, optionally, stabilizers. In soft capsules, theactive agents can be dissolved or suspended in suitable liquids, such asfatty oils, liquid paraffin, or liquid polyethylene glycol with orwithout stabilizers.

Aqueous suspensions can contain an active agent (e.g., a compositionused to practice the methods of the invention) in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethylene oxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol (e.g.,polyoxyethylene sorbitol mono-oleate), or a condensation product ofethylene oxide with a partial ester derived from fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). Theaqueous suspension can also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Formulations can be adjusted forosmolarity.

Oil-based pharmaceuticals are particularly useful for administrationhydrophobic active agents used to practice the methods of the invention.Oil-based suspensions can be formulated by suspending an active agent ina vegetable oil, such as arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin; or a mixture of these.See e.g., U.S. Pat. No. 5,716,928 describing using essential oils oressential oil components for increasing bioavailability and reducinginter- and intra-individual variability of orally administeredhydrophobic pharmaceutical compounds (see also U.S. Pat. No. 5,858,401).The oil suspensions can contain a thickening agent, such as beeswax,hard paraffin or cetyl alcohol. Sweetening agents can be added toprovide a palatable oral preparation, such as glycerol, sorbitol orsucrose. These formulations can be preserved by the addition of anantioxidant such as ascorbic acid. As an example of an injectable oilvehicle, see Minto (1997) J. Pharmacol. Exp. Ther. 281:93-102. Thepharmaceutical formulations of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil, described above, or a mixture of these. Suitableemulsifying agents include naturally-occurring gums, such as gum acaciaand gum tragacanth, naturally occurring phosphatides, such as soybeanlecithin, esters or partial esters derived from fatty acids and hexitolanhydrides, such as sorbitan mono-oleate, and condensation products ofthese partial esters with ethylene oxide, such as polyoxyethylenesorbitan mono-oleate. The emulsion can also contain sweetening agentsand flavoring agents, as in the formulation of syrups and elixirs. Suchformulations can also contain a demulcent, a preservative, or a coloringagent.

In practicing this invention, the pharmaceutical compounds can also beadministered by in intranasal, intraocular and intravaginal routesincluding suppositories, insufflation, powders and aerosol formulations(for examples of steroid inhalants, see Rohatagi (1995) J. Clin.Pharmacol. 35:1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol.75:107-111). Suppositories formulations can be prepared by mixing thedrug with a suitable non-irritating excipient which is solid at ordinarytemperatures but liquid at body temperatures and will therefore melt inthe body to release the drug. Such materials are cocoa butter andpolyethylene glycols.

In practicing this invention, the pharmaceutical compounds can bedelivered by transdermally, by a topical route, formulated as applicatorsticks, solutions, suspensions, emulsions, gels, creams, ointments,pastes, jellies, paints, powders, and aerosols.

In practicing this invention, the pharmaceutical compounds can also bedelivered as microspheres for slow release in the body. For example,microspheres can be administered via intradermal injection of drug whichslowly release subcutaneously; see Rao (1995) J. Biomater Sci. Polym.Ed. 7:623-645; as biodegradable and injectable gel formulations, see,e.g., Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres fororal administration, see, e.g., Eyles (1997) J. Pharm. Pharmacol.49:669-674.

In practicing this invention, the pharmaceutical compounds can beparenterally administered, such as by intravenous (IV) administration oradministration into a body cavity or lumen of an organ. Theseformulations can comprise a solution of active agent dissolved in apharmaceutically acceptable carrier. Acceptable vehicles and solventsthat can be employed are water and Ringer's solution, an isotonic sodiumchloride. In addition, sterile fixed oils can be employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid can likewise be used in the preparation ofinjectables. These solutions are sterile and generally free ofundesirable matter. These formulations may be sterilized byconventional, well known sterilization techniques. The formulations maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents, e.g., sodium acetate, sodiumchloride, potassium chloride, calcium chloride, sodium lactate and thelike. The concentration of active agent in these formulations can varywidely, and will be selected primarily based on fluid volumes,viscosities, body weight, and the like, in accordance with theparticular mode of administration selected and the patient's needs. ForIV administration, the formulation can be a sterile injectablepreparation, such as a sterile injectable aqueous or oleaginoussuspension. This suspension can be formulated using those suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation can also be a suspension in a nontoxicparenterally-acceptable diluent or solvent, such as a solution of1,3-butanediol. The administration can be by bolus or continuousinfusion (e.g., substantially uninterrupted introduction into a bloodvessel for a specified period of time).

The pharmaceutical compounds and formulations used to practice themethods of the invention can be lyophilized. The invention provides astable lyophilized formulation comprising a composition of theinvention, which can be made by lyophilizing a solution comprising apharmaceutical of the invention and a bulking agent, e.g., mannitol,trehalose, raffinose, and sucrose or mixtures thereof. A process forpreparing a stable lyophilized formulation can include lyophilizing asolution about 2.5 mg/mL protein, about 15 mg/mL sucrose, about 19 mg/mLNaCl, and a sodium citrate buffer having a pH greater than 5.5 but lessthan 6.5. See, e.g., U.S. patent app. no. 20040028670.

The compositions and formulations used to practice the methods of theinvention can be delivered by the use of liposomes (see also discussion,below). By using liposomes, particularly where the liposome surfacecarries ligands specific for target cells, or are otherwisepreferentially directed to a specific organ, one can focus the deliveryof the active agent into target cells in vivo. See, e.g., U.S. Pat. Nos.6,063,400; 6,007,839; Al-Muhammed (1996) J. Microencapsul. 13:293-306;Chonn (1995) Curr. Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J.Hosp. Pharm. 46:1576-1587.

The formulations used to practice the methods of the invention can beadministered for prophylactic and/or therapeutic treatments. Intherapeutic applications, compositions are administered to a subjectalready suffering from a condition, infection or disease in an amountsufficient to cure, alleviate or partially arrest the clinicalmanifestations of the condition, infection or disease and itscomplications (a “therapeutically effective amount”). For example, inalternative embodiments, pharmaceutical compositions of the inventionare administered in an amount sufficient to treat, prevent and/orameliorate normal, dysfunction (e.g., abnormally proliferating) cell,e.g., cancer cell, or blood vessel cell, including endothelial and/orcapillary cell growth; including neovasculature related to (within,providing a blood supply to) hyperplastic tissue, a granuloma or atumor. The amount of pharmaceutical composition adequate to accomplishthis is defined as a “therapeutically effective dose.” The dosageschedule and amounts effective for this use, i.e., the “dosing regimen,”will depend upon a variety of factors, including the stage of thedisease or condition, the severity of the disease or condition, thegeneral state of the patient's health, the patient's physical status,age and the like. In calculating the dosage regimen for a patient, themode of administration also is taken into consideration.

The dosage regimen also takes into consideration pharmacokineticsparameters well known in the art, i.e., the active agents' rate ofabsorption, bioavailability, metabolism, clearance, and the like (see,e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617;Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995)Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108;the latest Remington's, supra). The state of the art allows theclinician to determine the dosage regimen for each individual patient,active agent and disease or condition treated. Guidelines provided forsimilar compositions used as pharmaceuticals can be used as guidance todetermine the dosage regiment, i.e., dose schedule and dosage levels,administered practicing the methods of the invention are correct andappropriate.

Single or multiple administrations of formulations can be givendepending on the dosage and frequency as required and tolerated by thepatient. The formulations should provide a sufficient quantity of activeagent to effectively treat, prevent or ameliorate a conditions, diseasesor symptoms as described herein. For example, an exemplarypharmaceutical formulation for oral administration of compositions usedto practice the methods of the invention can be in a daily amount ofbetween about 0.1 to 0.5 to about 20, 50, 100 or 1000 or more ug perkilogram of body weight per day. In an alternative embodiment, dosagesare from about 1 mg to about 4 mg per kg of body weight per patient perday are used. Lower dosages can be used, in contrast to administrationorally, into the blood stream, into a body cavity or into a lumen of anorgan. Substantially higher dosages can be used in topical or oraladministration or administering by powders, spray or inhalation. Actualmethods for preparing parenterally or non-parenterally administrableformulations will be known or apparent to those skilled in the art andare described in more detail in such publications as Remington's, supra.

The methods of the invention can further comprise co-administration withother drugs or pharmaceuticals, e.g., compositions for treating cancer,septic shock, infection, fever, pain and related symptoms or conditions.For example, the methods and/or compositions and formulations of theinvention can be co-formulated with and/or co-administered withantibiotics (e.g., antibacterial or bacteriostatic peptides orproteins), particularly those effective against gram negative bacteria,fluids, cytokines, immunoregulatory agents, anti-inflammatory agents,complement activating agents, such as peptides or proteins comprisingcollagen-like domains or fibrinogen-like domains (e.g., a ficolin),carbohydrate-binding domains, and the like and combinations thereof.

Nanoparticles and Liposomes

The invention also provides nanoparticles and liposomal membranescomprising compounds used to practice the methods of the invention. Inalternative embodiments, the invention provides nanoparticles andliposomal membranes targeting diseased and/or tumor (cancer) stem cellsand dysfunctional stem cells, and angiogenic cells.

In alternative embodiments, the invention provides nanoparticles andliposomal membranes comprising (in addition to comprising compounds usedto practice the methods of the invention) molecules, e.g., peptides orantibodies, that selectively target abnormally growing, diseased,infected, dysfunctional and/or cancer (tumor) cell receptors. Inalternative embodiments, the invention provides nanoparticles andliposomal membranes using IL-11 receptor and/or the GRP78 receptor totargeted receptors on cells, e.g., on tumor cells, e.g., on prostate orovarian cancer cells. See, e.g., U.S. patent application publication no.20060239968.

In one aspect, the compositions used to practice the methods of theinvention are specifically targeted for inhibiting, ameliorating and/orpreventing endothelial cell migration and for inhibiting angiogenesis,e.g., tumor-associated or disease- or infection-associatedneovasculature.

The invention also provides nanocells to allow the sequential deliveryof two different therapeutic agents with different modes of action ordifferent pharmacokinetics, at least one of which comprises acomposition used to practice the methods of the invention. A nanocell isformed by encapsulating a nanocore with a first agent inside a lipidvesicle containing a second agent; see, e.g., Sengupta, et al., U.S.Pat. Pub. No. 20050266067. The agent in the outer lipid compartment isreleased first and may exert its effect before the agent in the nanocoreis released. The nanocell delivery system may be formulated in anypharmaceutical composition for delivery to patients suffering from adiseases or condition as described herein, e.g., such as a retinalage-related macular degeneration, a diabetic retinopathy, a cancer orcarcinoma, a glioblastoma, a neuroma, a neuroblastoma, a coloncarcinoma, a hemangioma, an infection and/or a condition with at leastone inflammatory component, and/or any infectious or inflammatorydisease, such as a rheumatoid arthritis, a psoriasis, a fibrosis,leprosy, multiple sclerosis, inflammatory bowel disease, or ulcerativecolitis or Crohn's disease.

In treating cancer, a traditional antineoplastic agent is contained inthe outer lipid vesicle of the nanocell, and an antiangiogenic agent ofthis invention is loaded into the nanocore. This arrangement allows theantineoplastic agent to be released first and delivered to the tumorbefore the tumor's blood supply is cut off by the composition of thisinvention.

The invention also provides multilayered liposomes comprising compoundsused to practice this invention, e.g., for transdermal absorption, e.g.,as described in Park, et al., U.S. Pat. Pub. No. 20070082042. Themultilayered liposomes can be prepared using a mixture of oil-phasecomponents comprising squalane, sterols, ceramides, neutral lipids oroils, fatty acids and lecithins, to about 200 to 5000 nm in particlesize, to entrap a composition of this invention.

A multilayered liposome used to practice the invention may furtherinclude an antiseptic, an antioxidant, a stabilizer, a thickener, andthe like to improve stability. Synthetic and natural antiseptics can beused, e.g., in an amount of 0.01% to 20%. Antioxidants can be used,e.g., BHT, erysorbate, tocopherol, astaxanthin, vegetable flavonoid, andderivatives thereof, or a plant-derived antioxidizing substance. Astabilizer can be used to stabilize liposome structure, e.g., polyolsand sugars. Exemplary polyols include butylene glycol, polyethyleneglycol, propylene glycol, dipropylene glycol and ethyl carbitol;examples of sugars are trehalose, sucrose, mannitol, sorbitol andchitosan, or a monosaccharides or an oligosaccharides, or a highmolecular weight starch. A thickener can be used for improving thedispersion stability of constructed liposomes in water, e.g., a naturalthickener or an acrylamide, or a synthetic polymeric thickener.Exemplary thickeners include natural polymers, such as acacia gum,xanthan gum, gellan gum, locust bean gum and starch, cellulosederivatives, such as hydroxy ethylcellulose, hydroxypropyl cellulose andcarboxymethyl cellulose, synthetic polymers, such as polyacrylic acid,poly-acrylamide or polyvinylpyrollidone and polyvinylalcohol, andcopolymers thereof or cross-linked materials.

Liposomes can be made using any method, e.g., as described in Park, etal., U.S. Pat. Pub. No. 20070042031, including method of producing aliposome by encapsulating a therapeutic product comprising providing anaqueous solution in a first reservoir; providing an organic lipidsolution in a second reservoir, wherein one of the aqueous solution andthe organic lipid solution includes a therapeutic product; mixing theaqueous solution with said organic lipid solution in a first mixingregion to produce a liposome solution, wherein the organic lipidsolution mixes with said aqueous solution so as to substantiallyinstantaneously produce a liposome encapsulating the therapeuticproduct; and immediately thereafter mixing the liposome solution with abuffer solution to produce a diluted liposome solution.

The invention also provides nanoparticles comprising compounds used topractice this invention to deliver a composition of the invention as adrug-containing nanoparticles (e.g., a secondary nanoparticle), asdescribed, e.g., in U.S. Pat. Pub. No. 20070077286. In one embodiment,the invention provides nanoparticles comprising a fat-soluble drug ofthis invention or a fat-solubilized water-soluble drug to act with abivalent or trivalent metal salt.

Liposomes

The compositions and formulations used to practice the invention can bedelivered by the use of liposomes. By using liposomes, particularlywhere the liposome surface carries ligands specific for target cells, orare otherwise preferentially directed to a specific organ, one can focusthe delivery of the active agent into target cells in vivo. See, e.g.,U.S. Pat. Nos. 6,063,400; 6,007,839; Al-Muhammed (1996) J.Microencapsul. 13:293-306; Chonn (1995) Curr. Opin. Biotechnol.6:698-708; Ostro (1989) Am. J. Hosp. Pharm. 46:1576-1587. For example,in one embodiment, compositions and formulations used to practice theinvention are delivered by the use of liposomes having rigid lipidshaving head groups and hydrophobic tails, e.g., as using apolyethyleneglycol-linked lipid having a side chain matching at least aportion the lipid, as described e.g., in US Pat App Pub No. 20080089928.In another embodiment, compositions and formulations used to practicethe invention are delivered by the use of amphoteric liposomescomprising a mixture of lipids, e.g., a mixture comprising a cationicamphiphile, an anionic amphiphile and/or neutral amphiphiles, asdescribed e.g., in US Pat App Pub No. 20080088046, or 20080031937. Inanother embodiment, compositions and formulations used to practice theinvention are delivered by the use of liposomes comprising apolyalkylene glycol moiety bonded through a thioether group and anantibody also bonded through a thioether group to the liposome, asdescribed e.g., in US Pat App Pub No. 20080014255. In anotherembodiment, compositions and formulations used to practice the inventionare delivered by the use of liposomes comprising glycerides,glycerophospholipides, glycerophosphinolipids, glycerophosphonolipids,sulfolipids, sphingolipids, phospholipids, isoprenolides, steroids,stearines, sterols and/or carbohydrate containing lipids, as describede.g., in US Pat App Pub No. 20070148220.

Antibodies as Pharmaceutical Compositions

In alternative embodiments, the invention provides compositions andmethods for inhibiting or depleting an integrin α_(v)β₃ (anb3), orinhibiting an integrin α_(v)β₃ (anb3) protein activity, or inhibitingthe formation or activity of an integrin anb3/RalB signaling complex, orinhibiting the formation or signaling activity of an integrin α_(v)β₃(anb3)/RalB/NFkB signaling axis; or inhibiting or depleting a RalBprotein or an inhibitor of RalB protein activation; or inhibiting ordepleting a Src or TBK1 protein or an inhibitor of Src or TBK1 proteinactivation. In alternative embodiments, this is achieved byadministration of inhibitory antibodies. For example, in alternativeembodiments, the invention uses isolated, synthetic or recombinantantibodies that specifically bind to and inhibit an integrin α_(v)β₃(anb3), or any protein of an integrin α_(v)β₃ (anb3)/RalB/NFkB signalingaxis, a RalB protein, a Src or TBK1 protein, or an NFkB protein.

In alternative aspects, an antibody for practicing the invention cancomprise a peptide or polypeptide derived from, modeled after orsubstantially encoded by an immunoglobulin gene or immunoglobulin genes,or fragments thereof, capable of specifically binding an antigen orepitope, see, e.g. Fundamental Immunology, Third Edition, W. E. Paul,ed., Raven Press, N.Y. (1993); Wilson (1994) J. Immunol. Methods175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97. Inalternative aspects, an antibody for practicing the invention includesantigen-binding portions, i.e., “antigen binding sites,” (e.g.,fragments, subsequences, complementarity determining regions (CDRs))that retain capacity to bind antigen, including (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR). Singlechain antibodies are also included by reference in the term “antibody.”

In alternative embodiments, the invention uses “humanized” antibodies,including forms of non-human (e.g., murine) antibodies that are chimericantibodies comprising minimal sequence (e.g., the antigen bindingfragment) derived from non-human immunoglobulin. In alternativeembodiments, humanized antibodies are human immunoglobulins in whichresidues from a hypervariable region (HVR) of a recipient (e.g., a humanantibody sequence) are replaced by residues from a hypervariable region(HVR) of a non-human species (donor antibody) such as mouse, rat, rabbitor nonhuman primate having the desired specificity, affinity, andcapacity. In alternative embodiments, framework region (FR) residues ofthe human immunoglobulin are replaced by corresponding non-humanresidues to improve antigen binding affinity.

In alternative embodiments, humanized antibodies may comprise residuesthat are not found in the recipient antibody or the donor antibody.These modifications may be made to improve antibody affinity orfunctional activity. In alternative embodiments, the humanized antibodycan comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the hypervariableregions correspond to those of a non-human immunoglobulin and all orsubstantially all of Ab framework regions are those of a humanimmunoglobulin sequence.

In alternative embodiments, a humanized antibody used to practice thisinvention can comprise at least a portion of an immunoglobulin constantregion (Fc), typically that of or derived from a human immunoglobulin.

However, in alternative embodiments, completely human antibodies alsocan be used to practice this invention, including human antibodiescomprising amino acid sequence which corresponds to that of an antibodyproduced by a human. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen bindingresidues.

In alternative embodiments, antibodies used to practice this inventioncomprise “affinity matured” antibodies, e.g., antibodies comprising withone or more alterations in one or more hypervariable regions whichresult in an improvement in the affinity of the antibody for antigen;e.g., NFkB, an integrin α_(v)β₃ (anb3), or any protein of an integrinα_(v)β₃ (anb3)/RalB/NFkB signaling axis, a RalB protein, a Src or TBK1protein, compared to a parent antibody which does not possess thosealteration(s). In alternative embodiments, antibodies used to practicethis invention are matured antibodies having nanomolar or even picomolaraffinities for the target antigen, e.g., NFkB, an integrin α_(v)β₃(anb3), or any protein of an integrin α_(v)β₃ (anb3)/RalB/NFkB signalingaxis, a RalB protein, a Src or TBK1 protein. Affinity matured antibodiescan be produced by procedures known in the art.

Antisense, siRNAs and microRNAs as Pharmaceutical Compositions

In alternative embodiments, the invention provides compositions andmethods for inhibiting or depleting an integrin α_(v)β₃ (anb3), orinhibiting an integrin α_(v)β₃ (anb3) protein activity, or inhibitingthe formation or activity of an integrin anb3/RalB signaling complex, orinhibiting the formation or signaling activity of an integrin α_(v)β₃(anb3)/RalB/NFkB signaling axis; or inhibiting or depleting a RalBprotein or an inhibitor of RalB protein activation; or inhibiting ordepleting a Src or TBK1 protein or an inhibitor of Src or TBK1 proteinactivation. In alternative embodiments, this is achieved byadministration of inhibitory nucleic acids, e.g., siRNA, antisensenucleic acids, and/or inhibitory microRNAs.

In alternative embodiments, compositions used to practice the inventionare formulated with a pharmaceutically acceptable carrier. Inalternative embodiments, the pharmaceutical compositions used topractice the invention can be administered parenterally, topically,orally or by local administration, such as by aerosol or transdermally.The pharmaceutical compositions can be formulated in any way and can beadministered in a variety of unit dosage forms depending upon thecondition or disease and the degree of illness, the general medicalcondition of each patient, the resulting preferred method ofadministration and the like. Details on techniques for formulation andadministration are well described in the scientific and patentliterature, see, e.g., the latest edition of Remington's PharmaceuticalSciences, Maack Publishing Co, Easton Pa. (“Remington's”).

While the invention is not limited by any particular mechanism ofaction: microRNAs (miRNAs) are short (20-24 nt) non-coding RNAs that areinvolved in post-transcriptional regulation of gene expression inmulticellular organisms by affecting both the stability and translationof mRNAs. miRNAs are transcribed by RNA polymerase II as part of cappedand polyadenylated primary transcripts (pri-miRNAs) that can be eitherprotein-coding or non-coding. The primary transcript is cleaved by theDrosha ribonuclease III enzyme to produce an approximately 70-ntstem-loop precursor miRNA (pre-miRNA), which is further cleaved by thecytoplasmic Dicer ribonuclease to generate the mature miRNA andantisense miRNA star (miRNA*) products. The mature miRNA is incorporatedinto a RNA-induced silencing complex (RISC), which recognizes targetmRNAs through imperfect base pairing with the miRNA and most commonlyresults in translational inhibition or destabilization of the targetmRNA.

In alternative embodiments pharmaceutical compositions used to practicethe invention are administered in the form of a dosage unit, e.g., atablet, capsule, bolus, spray. In alternative embodiments,pharmaceutical compositions comprise a compound, e.g., an antisensenucleic acid, e.g., an siRNA or a microRNA, in a dose: e.g., 25 mg, 30mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg,130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg,175 mg, 180 mg, 185 mg, 190 mg, 195 mg, 200 mg, 205 mg, 210 mg, 215 mg,220 mg, 225 mg, 230 mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg, 260 mg,265 mg, 270 mg, 270 mg, 280 mg, 285 mg, 290 mg, 295 mg, 300 mg, 305 mg,310 mg, 315 mg, 320 mg, 325 mg, 330 mg, 335 mg, 340 mg, 345 mg, 350 mg,355 mg, 360 mg, 365 mg, 370 mg, 375 mg, 380 mg, 385 mg, 390 mg, 395 mg,400 mg, 405 mg, 410 mg, 415 mg, 420 mg, 425 mg, 430 mg, 435 mg, 440 mg,445 mg, 450 mg, 455 mg, 460 mg, 465 mg, 470 mg, 475 mg, 480 mg, 485 mg,490 mg, 495 mg, 500 mg, 505 mg, 510 mg, 515 mg, 520 mg, 525 mg, 530 mg,535 mg, 540 mg, 545 mg, 550 mg, 555 mg, 560 mg, 565 mg, 570 mg, 575 mg,580 mg, 585 mg, 590 mg, 595 mg, 600 mg, 605 mg, 610 mg, 615 mg, 620 mg,625 mg, 630 mg, 635 mg, 640 mg, 645 mg, 650 mg, 655 mg, 660 mg, 665 mg,670 mg, 675 mg, 680 mg, 685 mg, 690 mg, 695 mg, 700 mg, 705 mg, 710 mg,715 mg, 720 mg, 725 mg, 730 mg, 735 mg, 740 mg, 745 mg, 750 mg, 755 mg,760 mg, 765 mg, 770 mg, 775 mg, 780 mg, 785 mg, 790 mg, 795 mg, or 800mg or more.

In alternative embodiments, an siRNA or a microRNA used to practice theinvention is administered as a pharmaceutical agent, e.g., a sterileformulation, e.g., a lyophilized siRNA or microRNA that is reconstitutedwith a suitable diluent, e.g., sterile water for injection or sterilesaline for injection. In alternative embodiments the reconstitutedproduct is administered as a subcutaneous injection or as an intravenousinfusion after dilution into saline. In alternative embodiments thelyophilized drug product comprises siRNA or microRNA prepared in waterfor injection, or in saline for injection, adjusted to pH 7.0-9.0 withacid or base during preparation, and then lyophilized. In alternativeembodiments a lyophilized siRNA or microRNA of the invention is betweenabout 25 to 800 or more mg, or about 25, 50, 75, 100, 125, 150, 175,200, 225, 250, 275, 300, 325, 350, 375, 425, 450, 475, 500, 525, 550,575, 600, 625, 650, 675, 700, 725, 750, 775, and 800 mg of a siRNA ormicroRNA of the invention. The lyophilized siRNA or microRNA of theinvention can be packaged in a 2 mL Type I, clear glass vial (e.g.,ammonium sulfate-treated), e.g., stoppered with a bromobutyl rubberclosure and sealed with an aluminum overseal.

In alternative embodiments, the invention provides compositions andmethods comprising in vivo delivery of antisense nucleic acids, e.g.,siRNA or microRNAs. In practicing the invention, the antisense nucleicacids, siRNAs, or microRNAs can be modified, e.g., in alternativeembodiments, at least one nucleotide of antisense nucleic acid, e.g.,siRNA or microRNA, construct is modified, e.g., to improve itsresistance to nucleases, serum stability, target specificity, bloodsystem circulation, tissue distribution, tissue penetration, cellularuptake, potency, and/or cell-permeability of the polynucleotide. Inalternative embodiments, the antisense nucleic acid, siRNA or microRNAconstruct is unmodified. In other embodiments, at least one nucleotidein the antisense nucleic acid, siRNA or microRNA construct is modified.

In alternative embodiments, guide strand modifications are made toincrease nuclease stability, and/or lower interferon induction, withoutsignificantly decreasing antisense nucleic acid, siRNA or microRNAactivity (or no decrease in antisense nucleic acid, siRNA or microRNAactivity at all). In certain embodiments, the modified antisense nucleicacid, siRNA or microRNA constructs have improved stability in serumand/or cerebral spinal fluid compared to an unmodified structure havingthe same sequence.

In alternative embodiments, a modification includes a 2′-H or2′-modified ribose sugar at the second nucleotide from the 5′-end of theguide sequence. In alternative embodiments, the guide strand (e.g., atleast one of the two single-stranded polynucleotides) comprises a2′-O-alkyl or 2′-halo group, such as a 2′-O-methyl modified nucleotide,at the second nucleotide on the 5′-end of the guide strand, or, no othermodified nucleotides. In alternative embodiments, polynucleotideconstructs having such modification may have enhanced target specificityor reduced off-target silencing compared to a similar construct withoutthe 2′-O-methyl modification at the position.

In alternative embodiments, a second nucleotide is a second nucleotidefrom the 5′-end of the single-stranded polynucleotide. In alternativeembodiments, a “2′-modified ribose sugar” comprises ribose sugars thatdo not have a 2′-OH group. In alternative embodiments, a “2′-modifiedribose sugar” does not include 2′-deoxyribose (found in unmodifiedcanonical DNA nucleotides), although one or more DNA nucleotides may beincluded in the subject constructs (e.g., a single deoxyribonucleotide,or more than one deoxyribonucleotide in a stretch or scattered inseveral parts of the subject constructs). For example, the 2′-modifiedribose sugar may be 2′-O-alkyl nucleotides, 2′-deoxy-2′-fluoronucleotides, 2′-deoxy nucleotides, or combination thereof.

In alternative embodiments, an antisense nucleic acid, siRNA or microRNAconstruct used to practice the invention comprises one or more 5′-endmodifications, e.g., as described above, and can exhibit a significantly(e.g., at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90% or more) less “off-target” gene silencing whencompared to similar constructs without the specified 5′-endmodification, thus greatly improving the overall specificity of theantisense nucleic acid, siRNA or microRNA construct of the invention.

In alternative embodiments, an antisense nucleic acid, siRNA or microRNAconstruct to practice the invention comprises a guide strandmodification that further increase stability to nucleases, and/or lowersinterferon induction, without significantly decreasing activity (or nodecrease in microRNA activity at all). In alternative embodiments, the5′-stem sequence comprises a 2′-modified ribose sugar, such as2′-O-methyl modified nucleotide, at the second nucleotide on the 5′-endof the polynucleotide, or, no other modified nucleotides. In alternativeembodiments the hairpin structure having such modification has enhancedtarget specificity or reduced off-target silencing compared to a similarconstruct without the 2′-O-methyl modification at same position.

In alternative embodiments, the 2′-modified nucleotides are some or allof the pyrimidine nucleotides (e.g., C/U). Examples of 2′-O-alkylnucleotides include a 2′-O-methyl nucleotide, or a 2′-O-allylnucleotide. In alternative embodiments, the modification comprises a2′-O-methyl modification at alternative nucleotides, starting fromeither the first or the second nucleotide from the 5′-end. Inalternative embodiments, the modification comprises a 2′-O-methylmodification of one or more randomly selected pyrimidine nucleotides (Cor U). In alternative embodiments, the modification comprises a2′-O-methyl modification of one or more nucleotides within the loop.

In alternative embodiments, the modified nucleotides are modified on thesugar moiety, the base, and/or the phosphodiester linkage. Inalternative embodiments the modification comprise a phosphate analog, ora phosphorothioate linkage; and the phosphorothioate linkage can belimited to one or more nucleotides within the loop, a 5′-overhang,and/or a 3′-overhang.

In alternative embodiments, the phosphorothioate linkage may be limitedto one or more nucleotides within the loop, and 1, 2, 3, 4, 5, or 6 morenucleotide(s) of the guide sequence within the double-stranded stemregion just 5′ to the loop. In alternative embodiments, the total numberof nucleotides having the phosphorothioate linkage may be about 12-14.In alternative embodiments, all nucleotides having the phosphorothioatelinkage are not contiguous. In alternative embodiments, the modificationcomprises a 2′-O-methyl modification, or, no more than 4 consecutivenucleotides are modified. In alternative embodiments, all nucleotides inthe 3′-end stem region are modified. In alternative embodiments, allnucleotides 3′ to the loop are modified.

In alternative embodiments, the 5′- or 3′-stem sequence comprises one ormore universal base-pairing nucleotides. In alternative embodimentsuniversal base-pairing nucleotides include extendable nucleotides thatcan be incorporated into a polynucleotide strand (either by chemicalsynthesis or by a polymerase), and pair with more than one pairing typeof specific canonical nucleotide. In alternative embodiments, theuniversal nucleotides pair with any specific nucleotide. In alternativeembodiments, the universal nucleotides pair with four pairings types ofspecific nucleotides or analogs thereof. In alternative embodiments, theuniversal nucleotides pair with three pairings types of specificnucleotides or analogs thereof. In alternative embodiments, theuniversal nucleotides pair with two pairings types of specificnucleotides or analogs thereof

In alternative embodiments, an antisense nucleic acid, siRNA or microRNAused to practice the invention comprises a modified nucleoside, e.g., asugar-modified nucleoside. In alternative embodiments, thesugar-modified nucleosides can further comprise a natural or modifiedheterocyclic base moiety and/or a natural or modified internucleosidelinkage; or can comprise modifications independent from the sugarmodification. In alternative embodiments, a sugar modified nucleoside isa 2′-modified nucleoside, wherein the sugar ring is modified at the 2′carbon from natural ribose or 2′-deoxy-ribose.

In alternative embodiments, a 2′-modified nucleoside has a bicyclicsugar moiety. In certain such embodiments, the bicyclic sugar moiety isa D sugar in the alpha configuration. In certain such embodiments, thebicyclic sugar moiety is a D sugar in the beta configuration. In certainsuch embodiments, the bicyclic sugar moiety is an L sugar in the alphaconfiguration. In alternative embodiments, the bicyclic sugar moiety isan L sugar in the beta configuration.

In alternative embodiments, the bicyclic sugar moiety comprises a bridgegroup between the 2′ and the 4′-carbon atoms. In alternativeembodiments, the bridge group comprises from 1 to 8 linked biradicalgroups. In alternative embodiments, the bicyclic sugar moiety comprisesfrom 1 to 4 linked biradical groups. In alternative embodiments, thebicyclic sugar moiety comprises 2 or 3 linked biradical groups.

In alternative embodiments, the bicyclic sugar moiety comprises 2 linkedbiradical groups. In alternative embodiments, a linked biradical groupis selected from —O—, —S—, —N(R1)-, —C(R1)(R₂)—, —C(R1)=C(R1)-,—C(R1)=N—, —C(═NR1)-, —Si(R1)(R₂)—, —S(═O)₂—, —S(═O)—, —C(═O)— and—C(═S)—; where each R1 and R₂ is, independently, H, hydroxyl, C1 to C₁₂alkyl, substituted C1-C12 alkyl, C₂-C12 alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C12 alkynyl, C₂-C20 aryl,substituted C₂-C20 aryl, a heterocycle radical, a substitutedheterocycle radical, heteroaryl, substituted heteroaryl, C₂-C₇ alicyclicradical, substituted C₂-C₇ alicyclic radical, halogen, substituted oxy(—O—), amino, substituted amino, azido, carboxyl, substituted carboxyl,acyl, substituted acyl, CN, thiol, substituted thiol, sulfonyl(S(═O)₂—H), substituted sulfonyl, sulfoxyl (S(═O)—H) or substitutedsulfoxyl; and each substituent group is, independently, halogen, C1-C₁₂alkyl, substituted C1-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, amino, substitutedamino, acyl, substituted acyl, C1-C₁₂ aminoalkyl, C1-C₁₂ aminoalkoxy,substituted C1-C₁₂ aminoalkyl, substituted C1-C₁₂ aminoalkoxy or aprotecting group.

In alternative embodiments, the bicyclic sugar moiety is bridged betweenthe 2′ and 4′ carbon atoms with a biradical group selected from—O—(CH₂)x-, —O—CH₂—, —O—CH₂CH₂—, —O—CH(alkyl)-, —NH—(CH2)P—,—N(alkyl)-(CH₂)x-, —O—CH(alkyl)-, —(CH(alkyl))-(CH2)x-, —NH—O—(CH2)x-,—N(alkyl)-O—(CH₂)x-, or —O—N(alkyl)-(CH₂)x-, wherein x is 1, 2, 3, 4 or5 and each alkyl group can be further substituted. In certainembodiments, x is 1, 2 or 3.

In alternative embodiments, a 2′-modified nucleoside comprises a2′-substituent group selected from halo, allyl, amino, azido, SH, CN,OCN, CF₃, OCF₃, O—, S—, or N(Rm)-alkyl; O—, S—, or N(Rm)-alkenyl; O—, S—or N(Rm)-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl,O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(Rm)(Rn) orO—CH2-C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently,

H, an amino protecting group or substituted or unsubstituted C1-C10alkyl. These 2′-substituent groups can be further substituted with oneor more substituent groups independently selected from hydroxyl, amino,alkoxy, carboxy, benzyl, phenyl, nitro (NO₂), thiol, thioalkoxy(S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl. In alternativeembodiments, a 2′-modified nucleoside comprises a 2′-substituent groupselected from F, O—CH₃, and OCH₂CH₂OCH₃.

In alternative embodiments, a sugar-modified nucleoside is a 4′-thiomodified nucleoside. In alternative embodiments, a sugar-modifiednucleoside is a 4′-thio-2′-modified nucleoside. In alternativeembodiments a 4′-thio modified nucleoside has a .beta.-D-ribonucleosidewhere the 4′-O replaced with 4′-S. A 4′-thio-2′-modified nucleoside is a4′-thio modified nucleoside having the 2′-OH replaced with a2′-substituent group. In alternative embodiments 2′-substituent groupsinclude 2′-OCH₃, 2′-O—(CH2)₂—OCH₃, and 2′-F.

In alternative embodiments, a modified oligonucleotide of the presentinvention comprises one or more internucleoside modifications. Inalternative embodiments, each internucleoside linkage of a modifiedoligonucleotide is a modified internucleoside linkage. In alternativeembodiments, a modified internucleoside linkage comprises a phosphorusatom.

In alternative embodiments, a modified antisense nucleic acid, siRNA ormicroRNA comprises at least one phosphorothioate internucleosidelinkage. In certain embodiments, each internucleoside linkage of amodified oligonucleotide is a phosphorothioate internucleoside linkage.

In alternative embodiments, a modified internucleoside linkage does notcomprise a phosphorus atom. In alternative embodiments, aninternucleoside linkage is formed by a short chain alkyl internucleosidelinkage. In alternative embodiments, an internucleoside linkage isformed by a cycloalkyl internucleoside linkages. In alternativeembodiments, an internucleoside linkage is formed by a mixed heteroatomand alkyl internucleoside linkage. In alternative embodiments, aninternucleoside linkage is formed by a mixed heteroatom and cycloalkylinternucleoside linkages. In alternative embodiments, an internucleosidelinkage is formed by one or more short chain heteroatomicinternucleoside linkages. In alternative embodiments, an internucleosidelinkage is formed by one or more heterocyclic internucleoside linkages.In alternative embodiments, an internucleoside linkage has an amidebackbone, or an internucleoside linkage has mixed N, O, S and CH2component parts.

In alternative embodiments, a modified oligonucleotide comprises one ormore modified nucleobases. In certain embodiments, a modifiedoligonucleotide comprises one or more 5-methylcytosines, or eachcytosine of a modified oligonucleotide comprises a 5-methylcytosine.

In alternative embodiments, a modified nucleobase comprises a5-hydroxymethyl cytosine, 7-deazaguanine or 7-deazaadenine, or amodified nucleobase comprises a 7-deaza-adenine, 7-deazaguanosine,2-aminopyridine or a 2-pyridone, or a modified nucleobase comprises a5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, or a 2 aminopropyladenine, 5-propynyluracil or a5-propynylcytosine.

In alternative embodiments, a modified nucleobase comprises a polycyclicheterocycle, or a tricyclic heterocycle; or, a modified nucleobasecomprises a phenoxazine derivative, or a phenoxazine further modified toform a nucleobase or G-clamp.

Therapeutically Effective Amount and Doses

In alternative embodiment, compounds, compositions, pharmaceuticalcompositions and formulations used to practice the invention can beadministered for prophylactic and/or therapeutic treatments; forexample, the invention provides compositions and methods for overcomingor diminishing or preventing Growth Factor Inhibitor (GFI) resistance ina cell, or, a method for increasing the growth-inhibiting effectivenessof a Growth Factor inhibitor on a cell, or, a method for re-sensitizinga cell to a Growth Factor Inhibitor. In alternative embodiments, theinvention provides compositions and methods for treating, preventing orameliorating: a disease or condition associated with dysfunctional stemcells or cancer stem cells, a retinal age-related macular degeneration,a diabetic retinopathy, a cancer or carcinoma, a glioblastoma, aneuroma, a neuroblastoma, a colon carcinoma, a hemangioma, an infectionand/or a condition with at least one inflammatory component, and/or anyinfectious or inflammatory disease, such as a rheumatoid arthritis, apsoriasis, a fibrosis, leprosy, multiple sclerosis, inflammatory boweldisease, or ulcerative colitis or Crohn's disease. In therapeuticapplications, compositions are administered to a subject alreadysuffering from a condition, infection or disease in an amount sufficientto cure, alleviate or partially arrest the clinical manifestations ofthe condition, infection or disease (e.g., disease or conditionassociated with dysfunctional stem cells or cancer stem cells) and itscomplications (a “therapeutically effective amount”). In the methods ofthe invention, a pharmaceutical composition is administered in an amountsufficient to treat (e.g., ameliorate) or prevent a disease or conditionassociated with dysfunctional stem cells or cancer stem cells. Theamount of pharmaceutical composition adequate to accomplish this isdefined as a “therapeutically effective dose.” The dosage schedule andamounts effective for this use, i.e., the “dosing regimen,” will dependupon a variety of factors, including the stage of the disease orcondition, the severity of the disease or condition, the general stateof the patient's health, the patient's physical status, age and thelike. In calculating the dosage regimen for a patient, the mode ofadministration also is taken into consideration.

Kits and Instructions

The invention provides kits comprising compositions for practicing themethods of the invention, including instructions for use thereof. Inalternative embodiments, the invention provides kits, blister packages,lidded blisters or blister cards or packets, clamshells, trays or shrinkwraps comprising a combination of compounds. In alternative embodiments,the combination of compounds comprises:

(1) at least one compound comprising or consisting of:

-   -   (i) an inhibitor or depleter of integrin α_(v)β₃ (anb3), or an        inhibitor of integrin α_(v)β₃ (anb3) protein activity, or an        inhibitor of the formation or activity of an integrin anb3/RalB        signaling complex, or an inhibitor of the formation or signaling        activity of an integrin α_(v)β₃ (anb3)/RalB/NFkB signaling axis,    -   wherein optionally the inhibitor of integrin α_(v)β₃ protein        activity is an allosteric inhibitor of integrin α_(v)β₃ protein        activity;    -   (ii) an inhibitor or depleter of RalB protein or an inhibitor of        RalB protein activation,    -   wherein optionally the inhibitor of RalB protein activity is an        allosteric inhibitor of RalB protein activity;    -   (iii) an inhibitor or depleter of Src or a Tank Binding Kinase        (TBK1) protein or an inhibitor of Src or TBK1 protein        activation,    -   wherein optionally the inhibitor of the Src or the TBK1 protein        activity is an amlexanox (or        2-amino-7-isopropyl-5-oxo-5H-chromeno[2,3-b]pyridine-3-carboxylic        acid) or APHTHASOL™,    -   and optionally the inhibitor of the Src or the TBK1 protein        activity is an allosteric inhibitor of Src or TBK1 protein        activity;    -   (iv) an inhibitor or depleter of NFKB or IRF3 protein or an        inhibitor of RalB protein activation,    -   wherein optionally the inhibitor of NFKB or IRF3 protein        activity is an allosteric inhibitor of NFKB or IRF3 protein        activity; or    -   (v) any combination of (i) to (iv); and

(2) at least one Growth Factor Inhibitor.

In alternative embodiments, the kit further comprises instructions forpracticing a method of the invention.

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples.

EXAMPLES Example 1 Methods of the Invention are Effective forSensitizing and Re-Sensitizing Cancer Cells to Growth Factor Inhibitors

The data presented herein demonstrates the effectiveness of thecompositions and methods of the invention in sensitizing andre-sensitizing cancer cells, and cancer stem cells, to growth factorinhibitors, and validates this invention's therapeutic approach toovercome growth factor inhibitor, e.g., EGFR inhibitor, resistance for awide range of cancers. The data presented herein demonstrates thatgenetic and pharmacological inhibition of RalB or NF-κB was able tore-sensitize αvβ3-expressing tumors to EGFR inhibitors.

Resistance to epidermal growth factor receptor (EGFR) inhibitors hasemerged as a significant clinical problem in oncology owing to variousresistance mechanisms^(1,2). Since cancer stem cells have beenassociated with drug resistance³, we examined the expression ofstem/progenitor cell markers for breast, pancreas and colon tumor cellswith acquired resistance to EGFR inhibitors. We found that CD61 (β3integrin) was the one marker consistently upregulated on EGFR inhibitorresistant tumor cells. Moreover, integrin αvβ3 expression was markedlyenhanced in murine orthotopic lung and pancreas tumors following theiracquired resistance to systemically delivered EGFR inhibitors. In fact,αvβ3 was both necessary and sufficient to account for the tumor cellresistance to EGFR inhibitors and other growth factor receptorinhibitors but not cytotoxic drugs.

Mechanistically, in drug resistant tumors αvβ3 forms a complex with KRASvia the adaptor Galectin-3 resulting in recruitment of RalB andactivation of its effector TBK1/NF-κB, revealing a previouslyundescribed integrin-mediated pathway. Accordingly, genetic orpharmacological inhibition of Galectin-3, RalB or NF-κB was able tore-sensitize αvβ3-expressing tumors to EGFR inhibitors, demonstratingthe effectiveness of the compositions and methods of the invention andvalidating this invention's therapeutic approach to overcome EGFRinhibitor resistance for a wide range of cancers.

Despite some level of clinical success achieved with EGFR TyrosineKinase inhibitors (TKIs), intrinsic and acquired cellular resistancemechanisms limit their efficacy^(1,2,4). A number of resistancemechanisms have been identified, including KRAS and EGFR mutations,resulting in constitutive activation of the ERK pathway⁵⁻⁷. WhileKRAS-mediated ERK signaling is associated with resistance to EGFRinhibition, KRAS also induces PI3K and Ral activation leading to tumorcell survival and proliferation^(8,9).

Nevertheless, it is clear that treatment of tumors with EGFR inhibitorsappears to select for a cell population that remains insensitive to EGFRblockade^(1,2). Prolonged administration of tumors with EGFR TKIs alsoselects for cells characterized by a distinct array of membraneproteins, including cancer stem/progenitor cell markers known to beassociated with increased cell survival and metastasis¹⁰. While a numberof EGFR-inhibitor resistance mechanisms have been defined, it is notclear whether a single unifying mechanism might drive the resistance ofa broad range of cancers.

To investigate this, we exposed pancreatic (FG, Miapaca-2), breast(BT474, SKBR3 and MDAMB468) and colon (SW480) human tumor cell lines toincreasing concentrations of erlotinib or lapatinib for three weeks, toselect cell subpopulations that were at least 10-fold more resistant tothese targeted therapies than their parental counterparts. Parent orresistant cells were then evaluated for a panel of stem/progenitor cellmarkers previously identified to be upregulated in the most aggressivemetastatic tumor cells¹¹⁻¹³.

As expected, the expression of some of these markers was significantlyincreased in one or more of these resistant cell populations.Surprisingly, we observed that CD61 (integrin β3) was the one markerupregulated in all resistant cell lines tested, FIG. 1 a. The longercells were exposed to erlotinib the greater the expression level of αvβ3was observed, FIG. 1 b. These findings were extended in vivo as micebearing orthotopic FG pancreatic tumors with minimal integrin αvβ3evaluated following four weeks of erlotinib treatment showed a 10-foldincrease in αvβ3 expression, FIG. 1 c. Moreover, H441 human lungadenocarcinoma orthotopic tumors¹⁴ exposed to systemic erlotinibtreatment in vivo for 7-8 weeks developed resistance and a qualitativeincrease in integrin αvβ3 expression compared with vehicle-treatedtumors, see FIG. 1 d and FIG. 5 (Supplementary FIG. 1). Thus, exposureof histologically distinct tumor cells in vitro or in vivo to EGFRinhibitors selects for a tumor cell population expressing high levels ofαvβ3.

In addition to being expressed on a subpopulation of stem/progenitorcells during mammary development¹⁵, αvβ3 is a marker of the mostmalignant tumor cells in a wide range of cancers^(16,17). To determinewhether endogenous expression of integrin αvβ3 might predict tumor cellresistance to EGFR blockade, various breast, lung and pancreatic tumorcells were first screened for αvβ3 expression and then analyzed fortheir sensitivity to EGFR inhibitors (Supplementary Table 1).

TABLE 1 Seguin et al, Supplementary Table 1 KRAS mutation, integrin αvβ3expression and EGFR TKI sensitivity of cancer cell lines Mutatedintegrin αvβ3 EGFR TKI Cell line Origin KRAS expression sensitive PANC-1pancreas yes yes no FG pancreas yes no yes Mapaca-2 (MP2) pancreas yesno yes CAPAN-1 pancreas yes no yes XPA-1 pancreas no no yes CFPAC-1pancreas yes yes no A549 lung yes yes no SKBR3 breast no no yes MDAMB231breast yes yes no MDAMB468 breast no no yes (MDA468) BT474 breast no noyes BT20 breast no yes no T47D breast yes no yes SW480 colon yes no yes

In all cases, β3 expressing tumor cells were intrinsically moreresistant to EGFR blockade than β3-negative tumor cell lines (FIG. 1 e).In fact, αvβ3 was required for resistance to EGFR inhibitors, sinceknockdown of αvβ3 in PANC-1 cells resulted in a 10-fold increase intumor cell sensitivity to erlotinib (FIG. 1 f). Moreover, integrin αvβ3was sufficient to induce erlotinib resistance since ectopic expressionof αvβ3 in FG cells lacking this integrin dramatically increasederlotinib resistance both, in vitro and in orthotopic pancreatic tumorsafter systemic treatment in vivo (FIGS. 1 f and g).

Integrin αvβ3 not only promotes adhesion-dependent signaling viaactivation of focal adhesion kinase FAK¹⁶ but it can also activate aFAK-independent signaling cascade in the absence of integrin ligationthat is associated with increased survival and tumor metastasis¹⁷. Todetermine whether αvβ3 ligation was required for its causative role inerlotinib resistance, FG cells transfected with either WT β3 or aligation deficient mutant of the integrin (D119A)¹⁷ were treated witherlotinib. The same degree of erlotinib resistance was observed in cellsexpressing either the ligation competent or incompetent form of integrinαvβ3, see FIG. 6 a (Supplementary FIG. 2 a) indicating that expressionof αvβ3, even in the unligated state, was sufficient to induce tumorcell resistance to erlotinib.

Tumor cells with acquired resistance to one drug can often displayresistance to a wide range of drugs^(18,19). Therefore, we examinedwhether αvβ3 expression also promotes resistance to other growth factorinhibitors and/or cytotoxic agents. Interestingly, while αvβ3 expressionaccounted for EGFR inhibitor resistance, it also induced resistance tothe IGFR inhibitor OSI-906, yet failed to protect cells from theantimetabolite agent gemcitabine and the chemotherapeutic agentcisplatin, see FIG. 6 b and FIG. 6 c (Supplementary FIGS. 2 b and c).These results demonstrate that integrin αvβ3 accounts for tumor cellresistance to drugs that target growth factor receptor mediated pathwaysbut does not promote for a more general resistant phenotype to alldrugs, particularly those that induce cell cytotoxicity.

In some cases oncogenic KRAS has been associated with EGFR TKIsresistance²⁰, however, it remains unclear whether oncogenic KRAS is aprerequisite for EGFR resistance²¹. Thus, we examined the KRASmutational status in various tumor cell lines and found that KRASoncogenic status did not account for resistance to EGFR inhibitors(Supplementary Table 1). Nevertheless, knockdown of KRAS in αvβ3expressing cells rendered them sensitive to erlotinib while KRASknockdown in cells lacking αvβ3 had no such effect, see FIG. 6 a andFIG. 6 b, indicating that αvβ3 and KRAS function cooperatively topromote tumor cell resistance to erlotinib. Interestingly, even innon-adherent cells, αvβ3 colocalized with oncogenic KRAS in the plasmamembrane (FIG. 2 c) and could be co-precipitated in a complex with KRAS,see FIG. 6 d. This interaction was specific for KRAS, as αvβ3 was notfound to associate with N-, R- or H-RAS isoforms in these cells, seeFIG. 6 d and FIG. 7 a and FIG. 7 b (Supplementary FIGS. 3 a and b).Furthermore, in BXPC3 human pancreatic tumor cells expressing wildtypeKRAS, αvβ3 showed increased association with KRAS only after these cellswere stimulated with EGF, see FIG. 6 e. Previous studies have indicatedthat the KRAS interacting protein Galectin-3 can also couple tointegrins^(22,23). Therefore, we considered whether Galectin-3 mightserve as an adaptor facilitating an interaction between αvβ3 and KRAS inepithelial tumor cells. In PANC-1 cells with endogenous β3 expression,αvβ3, KRAS, and Galectin-3 co-localized to membrane clusters, see FIG. 8a and FIG. 8 b (Supplementary FIG. 4 a-b). Furthermore, knockdown ofeither β3 or Galectin-3 prevented the localization of KRAS to thesemembrane clusters or their co-immunoprecipitation, see FIG. 8(Supplementary FIG. 4).

KRAS promotes multiple effector pathways including those regulated byRAF, phosphatidylinositol-3-OH kinases (PI3Ks) and RalGEFs leading to avariety of cellular functions²⁴. To investigate whether one or more KRASeffector pathway(s) may contribute to integrin β3/KRAS-mediated tumorcell resistance to EGFR inhibitors, we individually knocked-down orinhibited each downstream RAS effector in cells expressing or lackingintegrin αvβ3. While suppression of AKT, ERK and RalA sensitized tumorcells to erlotinib, regardless of the αvβ3 expression status, see FIG. 9(Supplementary FIG. 5), knockdown of RalB selectively sensitized αvβ3expressing tumor cells to erlotinib, see FIG. 7 a and FIG. 10 a(Supplementary FIG. 6 a). This was relevant to pancreatic tumor growthin vivo since, knockdown of RalB re-sensitized αvβ3-expressingpancreatic orthotopic tumors to erlotinib in mice, see FIG. 7 b. Infact, expression of a constitutively active RalB (G23V) mutant inβ3-negative cells was sufficient to confer resistance to EGFRinhibition, see FIG. 7 c and FIG. 10 b (Supplementary FIG. 6 b).Furthermore, ectopic expression of αvβ3 enhanced RalB activity in tumorcells in a KRAS-dependent manner, see FIG. 7 d). Accordingly, integrinαvβ3 and RalB were co-localized in tumor cells, see FIG. 10 c(Supplementary FIG. 7) and in human breast and pancreatic cancerbiopsies, see FIG. 11 (Supplementary FIG. 8) and a strong correlationwas found between αvβ3 expression and Ral GTPase activity in patientsbiopsies suggesting the αvβ3/RalB signaling module is clinicallyrelevant, see FIG. 7 e. Together, these findings indicate that integrinαvβ3 promotes erlotinib resistance of cancer cells by complexing withKRAS and RalB resulting in RalB activation.

RalB, an effector of RAS has been shown to induce TBK1/NF-κB activationleading to enhanced tumor cell survival^(25,26). In addition, it hasbeen shown that NF-κB signaling is essential for KRAS-driven tumorgrowth and resistance to EGFR blockade²⁷⁻²⁹. This prompted us to askwhether αvβ3 could regulate NF-κB activity through RalB activation andthereby promote tumor cell resistance to EGFR targeted therapy. To testthis, tumor cells expressing or lacking integrin αvβ3 and/or RalB weregrown in the presence or absence of erlotinib and lysates of these cellswere analyzed for activated downstream effectors of RalB. We found thaterlotinib treatment of αvβ3 negative cells reduced levels ofphosphorylated TBK1 and NF-κB, whereas in in-positive cells theseeffectors remained activated unless RalB was depleted, see FIG. 4 a.NF-κB activity was sufficient to account for EGFR inhibitor resistancesince ectopically expressed a constitutively active NF-κB (S276D) inβ3-negative FG pancreatic tumor cells³⁰ conferred resistance to EGFRinhibition, see FIG. 4 b). Accordingly, genetic or pharmacologicalinhibition of NF-κB in β3-positive cells completely restored erlotinibsensitivity³¹, see FIGS. 4 c and d). These findings demonstrate thatRalB, the effector of the αvβ3/KRAS complex, promotes tumor cellresistance to EGFR targeted therapy via TBK1/NF-κB activation. Together,our studies describe a role for αvβ3 mediating resistance to EGFRinhibition via RalB activation and its downstream effector NF-κB,opening new avenues to target tumors that are resistant to EGFR targetedtherapy, see FIG. 4 e.

Recent studies have shown that, upon prolonged treatment with EGFRinhibitors, tumor cells develop alternative or compensatory pathways tosustain cell survival, leading to drug resistance^(1,32). Here we showthat integrin αvβ3 is specifically upregulated in histologicallydistinct tumors where it accounts for resistance to EGFR inhibition. Atpresent, it is not clear whether exposure to EGFR inhibitors may promoteincreased αvβ3 expression or whether these drugs simply eliminate cellslacking αvβ3 allowing the expansion of αvβ3-expressing tumor cells.Given that integrin αvβ3 is a marker of mammary stem cells¹⁵, it ispossible that acquired resistance to EGFR inhibitors selects for a tumorstem-like cell population^(3,33). While integrins can promote adhesiondependent cell survival and induce tumor progression¹⁶, here, we showthat integrin αvβ3, even in the unligated state, can drive tumor cellsurvival and resistance to EGFR blockade by interaction with KRAS. Thisaction leads to the recruitment and activation of RalB and itsdownstream signaling effector NF-κB. In fact, NF-κB inhibitionre-sensitizes αvβ3-bearing tumors to EGFR blockade. Taken together, ourfindings not only identify αvβ3 as a tumor cell marker of drugresistance but reveal that inhibitors of EGFR and NF-κB should providesynergistic activity against a broad range of cancers.

FIGURE LEGENDS

FIG. 1. Integrin αvβ3 expression promotes resistance to EGFR TKI.

(a) Flow cytometric quantification of cell surface markers after 3 weekstreatment with erlotinib (pancreatic and colon cancer cells) orlapatinib (breast cancer cells). (b) Flow cytometric analysis of αvβ3expression in FG and Miapaca-2 cells following erlotinib. Error barsrepresent s.d. (n=3 independent experiments). (c) Top,immunofluorescence staining of integrin αvβ3 in tissue specimensobtained from orthotopic pancreatic tumors treated with vehicle (n=3) orerlotinib (n=4). Scale bar, 50 μm. Bottom, Integrin αvβ3 expression wasquantified as ratio of integrin αvβ3 pixel area over nuclei pixel areausing Metamorph (*P=0.049 using Mann-Whitney U test). (d) Right,intensity (scale 0 to 3) of β3 expression in mouse orthotopic lungtumors treated with vehicle (n=8) or erlotinib (n=7). Left,immunohistochemical staining of β3. Scale bar, 100 μm. (**P=0.0012 usingMann-Whitney U test) (e) IC₅₀ for cells treated with erlotinib orlapatinib. (f) Tumor sphere formation assay to establish a dose-responsefor erlotinib. Error bars represent s.d. (n=3 independent experiments).(g) Orthotopic FG tumors (>1000 mm³; n=10 per treatment group) weretreated for 10 days with vehicle or erlotinib. Results are expressed as% tumor weight compared to vehicle control. *P<0.05 Immunoblot analysisfor tumor lysates after 10 days of erlotinib confirms suppressed EGFRphosphorylation.

FIG. 2. Integrin αvβ3 cooperates with KRAS to promote resistance to EGFRblockade.

(a-b) Tumor sphere formation assay of FG expressing (a) or lacking (b)integrin β3 depleted of KRAS (shKRAS) or not (shCTRL) and treated with adose response of erlotinib. Error bars represent s.d. (n=3 independentexperiments). (c) Confocal microscopy images of PANC-1 and FG-β3 cellsgrown in suspension. Cells are stained for integrin αvβ3 (green), KRAS(red), and DNA (TOPRO-3, blue). Scale bar, 10 m. Data are representativeof three independent experiments. (d) RAS activity assay performed inPANC-1 cells using GST-Rafl-RBD immunoprecipitation as described inMethods Immunoblot analysis of KRAS, NRAS, HRAS, RRAS, integrin β1 andintegrin β3. Data are representative of three independent experiments.(e) Immunoblot analysis of Integrin αvβ3 immunoprecipitates from BxPC-3β3-positive cells grown in suspension and untreated or treated with EGF50 ng/ml for 5 minutes. RAS activity was determined using a GST-Rafl-RBDimmunoprecipitation assay. Data are representative of three independentexperiments.

FIG. 3. RalB is a key modulator of integrin αvβ3-mediated EGFR TKIresistance.

(a) Tumor spheres formation assay of FG-β3 treated with non-silencing(shCTRL) or RalB-specific shRNA and exposed to a dose response oferlotinib. Error bars represent s.d. (n=3 independent experiments).Immunoblot analysis showing RalB knockdown. (b) Effects of depletion ofRalB on erlotinib sensitivity in β3-positive tumor in a pancreaticorthotopic tumor model. Established β3-positive tumors expressingnon-silencing (shCTRL) or RalB-specific shRNA (>1000 mm³; n=13 pertreatment group) were randomized and treated for 10 days with erlotinib.Results are expressed as % of tumor weight changes after erlotinibtreatment compared to control. *P<0.05, **P<0.01. Tumor images, averageweights+/−s.e are shown. (c) Tumor spheres formation assay of FG cellsectopically expressing vector control, WT RalB FLAG tagged constructs ora constitutively active RalB G23V FLAG tagged treated with erlotinib(0.5 μM). Error bars represent s.d. (n=3 independent experiments).*P<0.05, NS=not significant. Immunoblot analysis showing RalB WT andRalB G23 FLAG tagged constructs transfection efficiency. (d) RalBactivity was determined in FG, FG-β3 expressing non-silencing orKRAS-specific shRNA, by using a GST-RalBP1-RBD immunoprecipitation assayas described in Methods. Data are representative of three independentexperiments. (e) Right, overall active Ral immunohistochemical stainingintensity between β3 negative (n=15) and β3 positive (n=70) humantumors. Active Ral staining was compared between each group by Fisher'sexact test (*P<0.05, P=0.036, two-sided). Left, representativeimmunohistochemistry images of human tumor tissues stained with anintegrin β3-specific antibody and an active Ral antibody. Scale bar, 50μm.

FIG. 4. Integrin αvβ3/RalB complex leads to NF-μB activation andresistance to EGFR TKI.

Immunoblot analysis of FG, FG-β3 and FG-β3 stably expressingnon-silencing or RalB-specific ShRNA, grown in suspension and treatedwith erlotinib (0.5 μM). pTBK1 refers to phospho-S172 TBK1, p-p65 NF-κBrefers to phospho-p65 NF-κB S276, pFAK refers to phospho-FAK Tyr 861.Data are representative of three independent experiments. (b) Tumorspheres formation assay of FG cells ectopically expressing vectorcontrol, WT NF-κB FLAG tagged or constitutively active S276D NF-κB FLAGtagged constructs treated with erlotinib (0.5 μM). Error bars represents.d. (n=3 independent experiments). *P<0.05, **P<0.001, NS=notsignificant Immunoblot analysis showing NF-κB WT and S276D NF-κB FLAGtransfection efficiency. (c) Tumor spheres formation assay of FG-β3treating with non-silencing (shCTRL) or NF-κB-specific shRNA and exposedto erlotinib (0.5 μM). Error bars represent s.d. (n=3 independentexperiments). *P<0.05, NS=not significant. (d) Dose response in FG-β3cells treated with erlotinib (10 nM to 5 μM), lenalidomide (10 nM to 5μM) or a combination of erlotinib (10 nM to 5 μM) and lenalidomide (1μM). Error bars represent s.d. (n=3 independent experiments). *P<0.05,NS=not significant. (e) Model depicting the integrin αvβ3-mediated EGFRTKI resistance and conquering EGFR TKI resistance pathway and itsdownstream RalB and NF-κB effectors.

Methods

Compounds and Cell Culture.

Human pancreatic (FG, PANC-1, Miapaca-2 (MP2), CFPAC-1, XPA-1, CAPAN-1,BxPc3), breast (MDAMB231, MDAMB468 (MDA468), BT20, SKBR3, BT474), colon(SW480) and lung (A549, H441) cancer cell lines were grown in ATCCrecommended media supplemented with 10% fetal bovine serum, glutamineand non-essential amino acids. We obtained FG-β3, FG-D119A mutant andPANC-shβ3 cells as previously described¹⁷. Erlotinib, OSI-906,Gemcitabine and Lapatinib were purchased from Chemietek. Cisplatin wasgenerated from Sigma-Aldrich. Lenalidomide was purchased from LCLaboratories. We established acquired EGFR™ resistant cells by adding anincreasing concentration of erlotinib (50 nM to 15 μM) or lapatinib (10nM to 15 μM), daily in 3D culture in 0.8% methylcellulose.

Lentiviral Studies and Transfection.

Cells were transfected with vector control, WT, G23V RalB-FLAG, WT and

S276D NF-κB-FLAG using a lentiviral system. For knock-down experiments,cells were transfected with KRAS, RalA, RalB, AKT1, ERK1/2, p65 NF-κBsiRNA (Qiagen) using the lipofectamine reagent (Invitrogen) followingmanufacturer's protocol or transfected with shRNA (Open Biosystems)using a lentiviral system. Gene silencing was confirmed by immunoblotsanalysis.

Tumor Sphere Formation.

Tumor spheres formation assays were performed essentially as describedpreviously¹⁷. Briefly, cells were seeded at 1000 to 2000 cells per welland grown for 12 days to 3 weeks. Cells were treated with vehicle(DMSO), erlotinib (10 nM to 5 μM), lapatinib (10 nM to 5 μM),gemcitabine (0.001 nM to 5 μM), OSI-906 (10 nM to 5 μM), lenalidomide(10 nM to 5 μM), or cisplatin (10 nM to 5 μM), diluted in DMSO. Themedia was replaced with fresh inhibitor every day for erlotinib,lapatinib, lenalidomide and 3 times a week for cisplatin andgemcitabine. Colonies were stained with crystal violet and scored withan Olympus SZH10 microscope. Survival curves were generated at leastwith five concentration points.

Flow Cytometry.

200,000 cells, after drug or vehicle treatment, were washed with PBS andincubated for 20 minutes with the Live/Dead reagent (Invitrogen)according to the manufacturer's instruction, then, cells were fixed with4% paraformaldehyde for 15 min and blocked for 30 min with 2% BSA inPBS. Cells were stained with fluorescent-conjugated antibodies to CD61(LM609), CD44 (eBioscience), CD24 (eBioscience), CD34 (eBioscience),CD133 (Santa Cruz), CD56 (eBioscience), CD29 (P4C10) and CD49f(eBioscience). All antibodies were used at 1:100 dilutions, 30 minutesat 4° C. After washing several times with PBS, cells were analyzed byFACS.

Immunohistochemical Analysis.

Immunostaining was performed according to the manufacturer'srecommendations (Vector Labs) on 5 μM sections of paraffin-embeddedtumors from the orthotopic xenograft pancreas and lung cancer mousemodels¹⁴ or from a metastasis tissue array purchased from US Biomax(MET961). Antigen retrieval was performed in citrate buffer pH 6.0 at95° C. for 20 min. Sections were treated with 0.3% H₂O₂ for 30 min,blocked in normal goat serum, PBS-T for 30 min followed by Avidin-D andthen incubated overnight at 4° C. with primary antibodies againstintegrin β3 (Abeam) and active Ral (NewEast) diluted 1:100 and 1:200 inblocking solution. Tissue sections were washed and then incubated withbiotinylated secondary antibody (1:500, Jackson ImmunoResearch) inblocking solution for 1 h. Sections were washed and incubated withVectastain ABC (Vector Labs) for 30 min. Staining was developed using aNickel-enhanced diamino-benzidine reaction (Vector Labs) and sectionswere counter-stained with hematoxylin. Sections stained with integrin β3and active Ral were scored by a H-score according to the stainingintensity (SI) on a scale 0 to 3 within the whole tissue section.

Immunoprecipitation and Immunoblot Analysis.

Cells were lysed in either RIPA lysis buffer (50 mM Tris pH 7.4, 100 mMNaCL, 2 mM EDTA, 10% DOC, 10% Triton, 0.1% SDS) or Triton lysis buffer(50 mM Tris pH 7.5, 150 mN NaCl, 1 mM EDTA, 5 mM MgCl2, 10% Glycerol, 1%Triton) supplemented with complete protease and phosphatase inhibitormixtures (Roche) and centrifuged at 13,000 g for 10 min at 4° C. Proteinconcentration was determined by BCA assay. 500 μg to 1 mg of proteinwere immunoprecipitated with 3 μg of anti-integrin αvβ-3 (LM609)overnight at 4° C. following by capture with 25 μl of protein A/G(Pierce). Beads were washed five times, eluted in Laemmli buffer,resolved on NuPAGE 4-12% Bis-Tris Gel (Invitrogen) and immunoblottingwas performed with anti-integrin β3 (Santa Cruz), anti-RalB (CellSignaling Technology), anti KRAS (Santa Cruz). For immunoblot analysis,25 μg of protein was boiled in Laemmli buffer and resolved on 8% to 15%gel. The following antibodies were used: KRAS (Santa Cruz), NRAS (SantaCruz), RRAS (Santa Cruz), HRAS (Santa Cruz), phospho-S172 NAK/TBK1(Epitomics), TBK1 (Cell Signaling Technology), phospho-p65NF-κB S276(Cell Signaling Technology), p65NF-κB (Cell Signaling Technology), RalB(Cell Signaling Technology), phospho-EGFR (Cell Signaling Technology),EGFR (Cell Signaling Technology), FLAG (Sigma), phospho-FAK Tyr 861(Cell Signaling Technology), FAK (Santa Cruz), Galectin 3 (BioLegend)and Hsp90 (Santa Cruz).

Affinity Pull-Down Assays for Ras and Ral.

RAS and Ral activation assays were performed in accordance with themanufacturer's (Upstate) instruction. Briefly, cells were cultured insuspension for 3 h, lysed and protein concentration was determined. 10μg of Ral Assay Reagent (Ral BP1, agarose) or RAS assay reagent (Raf-1RBD, agarose) was added to 500 mg to 1 mg of total cell protein in MLBbuffer (Millipore). After 30 min of rocking at 4° C., the activated(GTP) forms of RAS/Ral bound to the agarose beads were collected bycentrifugation, washed, boiled in Laemmli buffer, and loaded on a 15%SDS-PAGE gel.

Immunofluorescence Microscopy.

Frozen sections from tumors from the orthotopic xenograft pancreascancer mouse model or from patients diagnosed with pancreas or breastcancers (as approved by the institutional Review Board at University ofCalifornia, San Diego) or tumor cell lines were fixed in cold acetone or4% paraformaldehyde for 15 min, permeabilized in PBS containing 0.1%Triton for 2 min and blocked for 1 h at room temperature with 2% BSA inPBS. Cells were stained with antibodies to integrin αvβ3 (LM609), RalB(Cell Signaling Technology), Galectin 3 (BioLegend), pFAK (CellSignaling Technology), NRAS (Santa Cruz), RRAS (Santa Cruz), HRAS (SantaCruz) and KRAS (Abgent). All primary antibodies were used at 1:100dilutions, overnight at 4° C. Where mouse antibodies were used on mousetissues, we used the MOM kit (Vector Laboratory). After washing severaltimes with PBS, cells were stained for two hours at 4° C. with secondaryantibodies specific for mouse or rabbit (Invitrogen), as appropriate,diluted 1:200 and co-incubated with the DNA dye TOPRO-3 (1:500)(Invitrogen). Samples were mounted in VECTASHIELD hard-set media (VectorLaboratories) and imaged on a Nikon Eclipse C1 confocal microscope with1.4 NA 60× oil-immersion lens, using minimum pinhole (30 m). Images werecaptured using 3.50 imaging software. Colocalization between Integrinαvβ3 and KRAS was studied using the Zenon Antibody Labeling Kits(Invitrogen).

Orthotopic Pancreas Cancer Xenograft Model.

All mouse experiments were carried out in accordance with approvedprotocols from the UCSD animal subjects committee and with theguidelines set forth in the NIH Guide for the Care and Use of LaboratoryAnimals. Tumors were generated by injection of FG human pancreaticcarcinoma cells (10⁶ tumor cells in 30 μL of sterile PBS) into the tailof the pancreas of 6-8 week old male immune compromised nu/nu mice.Tumors were established for 2-3 weeks (tumor sizes were monitored byultrasound) before beginning dosing. Mice were dosed by oral gavage withvehicle (6% Captisol) or 100 mg/kg/day erlotinib for 10 to 30 days priorto harvest.

Orthotopic Lung Cancer Xenograft Model.

Tumors were generated by injection of H441 human lung adenocarcinomacells (10⁶ tumor cells per mouse in 50 μL of HBSS containing 50 mggrowth factor-reduced Matrigel (BD Bioscience) into the left thorax atthe lateral dorsal axillary line and into the left lung, as previouslydescribed¹⁴ of 8 week old male immune-compromised nu/nu mice. 3 weeksafter tumor cell injection, the mice were treated with vehicle orerlotinib (100 mg/kg/day) by oral gavage until moribund (approximately50 and 58 days, respectively).

Statistical Analyses.

All statistical analyses were performed using Prism software (GraphPad).Two-tailed Mann Whitney U tests, Fisher's exact tests, or t-tests wereused to calculate statistical significance. A P value<0.05 wasconsidered to be significant.

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A senescence program controlled by p53    and p16INK4a contributes to the outcome of cancer therapy. Cell 109,    335-346 (2002).-   20. Baselga, J. & Rosen, N. Determinants of RASistance to    anti-epidermal growth factor receptor agents. J Clin Oncol 26,    1582-1584 (2008).-   21. Moore, M. J., et al. Erlotinib plus gemcitabine compared with    gemcitabine alone in patients with advanced pancreatic cancer: a    phase III trial of the National Cancer Institute of Canada Clinical    Trials Group. J Clin Oncol 25, 1960-1966 (2007).-   22. Levy, R., Grafi-Cohen, M., Kraiem, Z. & Kloog, Y. Galectin-3    promotes chronic activation of K-Ras and differentiation block in    malignant thyroid carcinomas. Molecular Cancer Therapeutics 9,    2208-2219 (2010).-   23. Markowska, A. I., Liu, F. T. & Panjwani, N. Galectin-3 is an    important mediator of VEGF- and bFGF-mediated angiogenic response.    The Journal of Experimental Medicine 207, 1981-1993 (2010).-   24. Buday, L. & Downward, J. 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Example 2 Methods of the Invention are Effective for Sensitizing andRe-Sensitizing Cancer Cells to Growth Factor Inhibitors

The data presented herein demonstrates the effectiveness of thecompositions and methods of the invention in sensitizing andre-sensitizing cancer cells, and cancer stem cells, to growth factorinhibitors, and validates this invention's therapeutic approach toovercome growth factor inhibitor resistance for a wide range of cancers.In particular, the data presented in this Example demonstrates that β3integrin induces erlotinib resistance in cancer cells by switching tumordependency from EGFR to KRAS.

In alternative embodiments, the compositions and methods of theinvention overcome tumor drug resistance that limits the long-termsuccess of therapies targeting EGFR. Here, we identify integrin αvβ3 asa biomarker of intrinsic and acquired resistance to erlotinib in humanpancreatic and lung carcinomas irrespective of their KRAS mutationalstatus. Functionally, αvβ3 is necessary and sufficient for thisresistance where it acts in the unligated state as a scaffold to recruitactive KRAS into membrane clusters switching tumor dependency from EGFRto KRAS. The KRAS effector RalB is recruited to this complex, where itmediates erlotinib resistance via a TBK-1/NF-κB pathway. Disruptingassembly of this complex or inhibition of its downstream effectors fullyrestores tumor sensitivity to EGFR blockade. Our findings uncouple KRASmutations from erlotinib resistance, revealing an unexpected requirementfor integrin αvβ3 in this process.

We hypothesized that upregulation of specific genes common to multipletumor types exposed to erlotinib drives a conserved pathway that governsboth intrinsic and acquired resistance. To identify genes associatedwith erlotinib (N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine) resistance, we analyzed the expression of a tumorprogression gene array for human cell lines with intrinsic resistance ormurine xenografts following the acquisition of resistance in vivo. Themost upregulated gene common to all drug resistant carcinomas tested wasthe cell surface ITGB3, integrin β3 (FIG. 1A, and table S1) associatedwith the integrin αvβ3 whose expression has been linked to tumorprogression. αvβ3 expression completely predicted erlotinib resistancefor a panel of histologically distinct tumor cell lines (FIG. 1B andFIG. S1B). Moreover, chronic treatment of the erlotinib sensitive linesresulted in the induction of β3 expression concomitantly with drugresistance (FIG. 1C and FIG. S1B, C). We also detected increased β3expression in lung carcinoma patients who had progressed on erlotinibtherapy (FIG. S2). In addition, we examined both treatment naive anderlotinib resistant NSCLC patients from the BATTLE Study (10) ofnon-small cell lung cancer (NSCLC) and found β3 gene expression wassignificantly higher in patients who progressed on erlotinib (FIG. 1D).Finally, we examined serial primary lung tumors biopsies from patientsbefore treatment or after erlotinib resistance and found a qualitativeincrease in integrin β3 expression concurrent with the loss of erlotinibsensitivity (FIG. 1E). Taken together, our findings show that integrinβ3 is a marker of acquired and intrinsic erlotinib resistance forpancreas and lung cancer.

To assess the functional role of αvβ3 in erlotinib resistance we used again and loss-of-function approach and found that integrin β3 was bothnecessary and sufficient to account for erlotinib resistance in vitroand during systemic treatment of lung and orthotopic pancreatic tumorsin vivo (FIG. 1F, G and FIG. S3A-C). Interestingly, integrin β3expression did not impact resistance to chemotherapeutic agents such asgemcitabine and cisplatin while conferring resistance to inhibitorstargeting EGFR1/EGFR2 or IGFR (FIG. S3C-E), suggesting this integrinplays a specific role in tumor cell resistance to RTK inhibitors.

As integrin αvβ3 is functions as an adhesion receptor, ligand bindinginhibitors could represent a therapeutic strategy to sensitize tumors toEGFR inhibitors. However, αvβ3 expression induced drug resistance incells growing in suspension. Also, neither function blocking antibodiesnor cyclic peptide inhibitors sensitized integrin αvβ3-expressing tumorsto EGFR inhibitors (not shown), and tumor cells expressing wild-typeintegrin β3 or the ligation-deficient mutant β3 D119A (11) showedequivalent drug resistance (FIG. S4). Since the contribution of integrinαvβ3 to erlotinib resistance appears to involve a non-canonical,ligation-independent mechanism that is not sensitive to traditionalintegrin antagonists, understanding the molecular mechanisms drivingthis pathway could provide therapeutic opportunities.

Integrins function in the context of RAS family members. Interestingly,we found that αvβ3 associated with KRAS but not N-, H- or R-RAS (FIG.2A). While oncogenic KRAS has been linked to erlotinib resistance, thereare many notable exceptions (6-9). In fact, we observed a number oftumor cell lines with oncogenic KRAS to be sensitive to erlotinib (FG,H441, and CAPAN1), whereas H1650 cells were erlotinib resistant despitetheir expression of wildtype KRAS and mutant EGFR (table S2). In fact,αvβ3 expression consistently correlated with erlotinib resistance forall cell lines tested (Pearson's correlation coefficient R²=0.87) makinga better predictor of erlotinib resistance. Interestingly, we observedactive KRAS to be distributed within the cytoplasm in β3-negative cells(FIG. S5A) whereas in cells expressing β3 endogenously or ectopically,KRAS was localized to β3-containing membrane clusters, even in thepresence of erlotinib (FIG. 2B,C and FIG. S5A) a relationship that wasnot observed for β1 integrin (FIGS. S5B and C). Furthermore, knockdownof KRAS impaired tumorsphere formation and restored erlotinibsensitivity in β3-positive cells (FIG. 2D-F and FIG. S6A-C). Incontrast, KRAS was dispensable for tumorsphere formation and erlotinibresponse the in cells lacking β3 expression (FIG. 2D-F). Thus, β3integrin expression switches tumor cell dependency from EGFR to KRAS,and that the localization of β3 with KRAS at the plasma membrane appearsto be a critical determinant of tumor cell resistance to erlotinib.Also, our results reveal that tumors expressing oncogenic KRAS withoutβ3 remain sensitive to EGFR blockade.

Independent studies have shown that galectin-3 can interact with eitherKRAS (12) or β3 (13) so we asked whether this protein might serve as anadaptor to promote KRAS/β3 complex formation. Underanchorage-independent growth conditions, integrin β3, KRAS, andGalectin-3 were co-localized in membrane clusters (FIG. 2G and FIG. S7),and knockdown of either integrin β3 or Galectin-3 prevented complexformation, KRAS membrane localization, and importantly sensitized αvβ3expressing tumors to erlotinib (FIG. 2G-I).

We next evaluated the signaling pathways driven by the integrin β3/KRAScomplex. Erlotinib resistance of β3-positive cells was not affected bydepletion of known KRAS effectors, including AKT, ERK, or RalA (FIG.S8A,B). However, knockdown of RalB sensitized β3-expressing cells toerlotinib in vitro (FIG. 3A and FIG. S8A-C) and in pancreatic orthotopictumors in vivo (FIG. 3B). Accordingly, expression of constitutivelyactive RalB in β3-negative cells conferred erlotinib resistance (FIG.3C). Mechanistically, RalB was recruited to the β3/KRAS membraneclusters (FIG. 3D-F) where it became activated in a KRAS-dependentmanner (FIG. 3G). Recent studies have reported that TBK1 and NF-κB areRalB effectors linked to KRAS dependency (14) and erlotinib resistance(15). We found that erlotinib decreased the activation of theseeffectors only in the absence of integrin β3 (FIG. 3H). In fact, loss ofRalB in β3-expressing cells restored erlotinib-mediated inhibition ofTBK1 and NF-κB (FIG. 3H). Accordingly, depletion of either TBK1 or NF-κBsensitized β3-positive cells to erlotinib (FIG. 3I and FIG. S9A), whileectopic expression of activated NF-κB was sufficient to promote drugresistance in β3-negative cells (FIG. S9B). To evaluate the therapeuticpotential of targeting this pathway, we examined whether erlotinibresistance of β3-expressing tumors could be reversed with approved drugsknown to suppress NF-κB activation, lenalidomide/REVLIMID®(16) andbortezomib/VELCADE® (17). While monotherapy with these drugs failed toimpact tumor growth, either drug used combination with erlotinibdecreased tumorsphere formation in vitro (FIG. 4A) and completelysuppressed tumor growth in vivo (FIG. 4B, C and FIG. S10). Thesefindings support the model depicted in FIG. 4D where inhibition of NF-κBrestores erlotinib sensitivity in β3 expressing tumors. These findingssupport the model depicted in FIG. 4D that αvβ3 expression in lung andpancreatic tumors recruits oncogenic KRAS facilitating NFκB activityleading to erlotinib resistance which can be overcome by a combinationof currently approved inhibitors of NF-κB and EGFR.

See also FIG. 40 and FIG. 41, graphically illustrating datademonstrating that depletion of RalB overcomes erlotinib resistance inKRAS mutant cells, and depletion of TBK1 overcomes erlotinib resistancein KRAS mutant cells, respectively. In FIG. 41: Integin b3 mediates TBK1activation through RalB and TBK1 depletion overcomes integrinb3-mediated erlotinib resistance.

Our observations demonstrate that the ability of β3 integrin to recruitKRAS into a membrane complex along with Galectin-3 and RalB functions toswitch tumor cell dependency from EGFR to KRAS. In fact, oncogenic KRASrequires this non-canonical β3-mediated pathway to drive erlotinibresistance. We show that currently available approved inhibitors of thispathway can be used to practice the methods of this invention to treatpatients with solid tumors, rendering them sensitive to EGFR inhibitorssuch as erlotinib.

Material and Methods

Compounds and cell culture. Human pancreatic (FG, PANC-1, CFPAC-1,XPA-1, HPAFII, CAPAN-1, BxPC3) and lung (A549, H441, HCC827 and H1650)cancer cell lines were grown in ATCC recommended media supplemented with10% fetal bovine serum, glutamine and non-essential amino acids. Weobtained FG-β3, FG-D119A mutant and PANC-shβ3 cells as previouslydescribed (10). Erlotinib, OSI-906, Gemcitabine, Bortezomib andLapatinib were purchased from Chemietek. Cisplatin was generated fromSigma-Aldrich. Lenalidomide was purchased from LC Laboratories. Geneexpression analysis. The Tumor Metastasis PCR Array (Applied Biosystem),consisting of 92 genes known to be involved in tumor progression andmetastasis, was used to profile the common genes upregulated inerlotinib-resistant cells compared to erlotinib-sensitive cellsaccording to the manufacturer's instructions. Briefly, total RNA wasextracted and reverse transcribed into cDNA using the RNeasy kit(Qiagen). The cDNA was combined with a SYBR Green qPCR Master Mix(Qiagen), and then added to each well of the same PCR Array plate thatcontained the predispensed gene-specific primer sets.Tumor digestion and Flow Cytometry. Fresh tumor tissue from lung cancercell lines was mechanically dissociated and then enzymatically digestedin trypsin. The tissue was further filtered through a cell strainer toobtain a suspension of single tumor cells. Then, cells were washed werewashed with PBS and incubated for 20 minutes with the Live/Dead reagent(Invitrogen) according to the manufacturer's instruction, then, cellswere fixed with 4% paraformaldehyde for 15 min and blocked for 30 minwith 2% BSA in PBS. Cells were stained with fluorescent-conjugatedantibodies to integrin αvβ3 (LM609, Cheresh Lab), After washing severaltimes with PBS, cells were analyzed by FACS.Tumorsphere assay. Tumorsphere assay was performed as previouslydescribed (10). Cells were treated with vehicle (DMSO), erlotinib (10 nMto 5 μM), lapatinib (10 nM to 5 μM), gemcitabine (0.001 nM to 5 μM),OSI-906 (10 nM to 5 μM), lenalidomide (1 μM), cisplatin (10 nM to 5 μM),or bortezomib (4 nM) diluted in DMSO. The media was replaced with freshinhibitor 2/6 times a week. Survival curves were generated at least withfive concentration points.

Mouse cancer models. All research was conducted under protocol S05018and approved by the University of California—San Diego InstitutionalAnimal Care and Use Committee (IACUC). FG pancreatic carcinoma cells(1×106 tumor cells in 30 μl of PBS) were injected into the pancreas of6- to 8-week-old male nude mice as previously described (10). Tumorswere established for 2-3 weeks (tumor sizes were monitored byultrasound) before beginning dosing. Mice were dosed by oral gavage withvehicle (6% Captisol) or 10, 25 and 50 mg/kg/day erlotinib for 10 to 30days prior to harvest. H441 lung adenocarcinoma cells were generated aspreviously described (21). 3 weeks after tumor cell injection, the micewere treated with vehicle or erlotinib (100 mg/kg/day) by oral Mousecancer models. All research was conducted under protocol S05018 andapproved by the University of California—San Diego Institutional AnimalCare and Use Committee (IACUC). FG pancreatic carcinoma cells (1×106tumor cells in 30 μl of PBS) were injected into the pancreas of 6- to8-week-old male nude mice as previously described (10). Tumors wereestablished for 2-3 weeks (tumor sizes were monitored by ultrasound)before beginning dosing. Mice were dosed by oral gavage with vehicle (6%Captisol) or 10, 25 and 50 mg/kg/day erlotinib for 10 to 30 days priorto harvest. H441 lung adenocarcinoma cells were generated as previouslydescribed (21). 3 weeks after tumor cell injection, the mice weretreated with vehicle or erlotinib (100 mg/kg/day) by oral gavage untilmoribund (approximately 50 and 58 days, respectively). To generatesubcutaneous tumors, FG-β3, FG-R (after erlotinib resistance) andHCC-827 human carcinoma cells (5×106 tumor cells in 200 μl of PBS) wereinjected subcutaneously to the left or right flank of 6-8-week-oldfemale nude mice. Tumors were measured every 2-3 days with calipersuntil they were harvested at day 10, 16 or after acquired resistance.

NSCLC specimens from the BATTLE trial. The BATTLE (Biomarker-integratedApproaches of Targeted Therapy for Lung Cancer Elimination) trial was arandomized phase II, single-center, open-label study in patients withadvanced NSCLC refractory to prior chemotherapy and included patientswith and without prior EGFR inhibitor treatment (12). Patients underwenta tumor new biopsy prior to initiating study treatment. The microarrayanalysis of mRNA expression on frozen tumor core biopsies was conductedusing the Affymetrix Human Gene 1.ST™ platform as previously described(22).Serial biopsies from NSCLC patients. Tumor biopsies from University ofCalifornia, San Diego (UCSD) Medical Center stage IV non-small cell lungcancer patients were obtained before erlotinib treatment and 3 patientsbefore and after erlotinib resistance. All biopsies are from lung orpleural effusion. Patients 1 had a core biopsy from the primary lungtumor, and Patient 2 and 3 had a fine needle biopsy from a pleuraleffusion. All patients had an initial partial response, followed bydisease progression after 920, 92, and 120 days of erlotinib therapy,respectively. This work was approved by the UCSD Institutional ReviewBoard (IRB).Immunofluorescence microscopy. Frozen sections from tumors fromorthotopic pancreatic tumors, from patients diagnosed with pancreascancers (as approved by the institutional Review Board at University ofCalifornia, San Diego) or tumor cell lines were processed as previouslydescribed (23). Cells were stained with indicated primary, followed bysecondary antibodies specific for mouse or rabbit (Invitrogen), asappropriate. Samples imaged on a Nikon ECLIPSE C1™ confocal microscopewith 1.4 NA 60× oil-immersion lens, using minimum pinhole (30 μm). Thefollowing antibodies were used: anti-integrin β3 (LM609), KRAS (Pierceand Abgent M01), Galectin-3, NRAS, RRAS,Genetic knockdown and expression of mutant constructs. Cells weretransfected with vector control, WT, G23V RalB-FLAG, WT and S276DNF-κB-FLAG using a lentiviral system. For knock-down experiments, cellswere transfected with a pool of RalA, RalB, AKT1, ERK1/2 siRNA (Qiagen)using the lipofectamine reagent (Invitrogen) following manufacturer'sprotocol or transfected with shRNA (integrin β3, KRAS, Galectin-3, RalB,TBK1 and p65NF-κB) (Open Biosystems) using a lentiviral system. Genesilencing was confirmed by immunoblots analysis.Immunohistochemical analysis Immunostaining was performed according tothe manufacturer's recommendations (Vector Labs) on 5 μM sections ofparaffin-embedded tumors from tumor biopsies from lung cancer patients.Tumor sections were processed as previously described (23) usingintegrin β3 (Abcam clone EP2417Y). Sections stained with integrin β3were scored by a H-score according to the staining intensity (SI) on ascale 0 to 3 within the whole tissue section.Immunoprecipitation and immunoblots. Lysates from cell lines andxenograft tumors were generated using standard methods and RIPA orTriton buffers.Immunoprecipitation experiments were performed as previously described(23) with anti-integrin αvβ3 (LM609) or Galectin-3. For immunoblotanalysis, 25 μg of protein was boiled in Laemmli buffer and resolved on8% to 15% gel. The following antibodies were used: anti-integrin β3,KRAS, NRAS, RRAS, HRAS, Hsp60 and Hsp90 from Santa Cruz, phospho-S172NAK/TBK1 from Epitomics, TBK1, phospho-p65NF-κB S276, p65NF-κB, RalB,phospho-EGFR, EGFR, from Cell Signaling Technology, and Galectin 3 fromBioLegend.Membrane extract. Membrane fraction from FG and FG-β3 grown insuspension in media complemented with 0.1% BSA were isolated using theMEM-PER membrane extraction kit (Fisher) according to the manufacturer'sinstructions. Affinity pull-down assays for Ras and Ral. RAS and Ralactivation assays were performed in accordance with the manufacturer's(Upstate) instruction. Briefly, cells were cultured in suspension for 3h. 10 μg of Ral Assay Reagent (Ral BP1, agarose) or RAS assay reagent(Raf-1 RBD, agarose) was added to 500 mg to 1 mg of total cell proteinin MLB buffer (Millipore). After 30 min of rocking at 40 C, theactivated (GTP) forms of RAS/Ral bound to the agarose beads werecollected by centrifugation, washed, boiled in Laemmli buffer, andloaded on a 15% SDS-PAGE gel.Statistical Analyses. All statistical analyses were performed usingPrism software (GRAPHPAD™). Two-tailed Mann Whitney U tests, Chi-squaredtests, one way ANOVA tests or t-tests were used to calculate statisticalsignificance. A P value<0.05 was considered to be significant.

FIGURE LEGENDS

FIG. 1 (FIG. 12/31) illustrates data showing that integrin β3 isexpressed in EGFR inhibitor resistant tumors and is necessary andsufficient to drive EGFR inhibitor resistance.

(A) Identification of the most upregulated tumor progression genescommon to erlotinib resistant carcinomas. (B) Erlotinib IC₅₀ in a panelof human carcinoma cell lines treated with erlotinib in 3D culture. n=3independent experiments. (C) Percentage of integrin β3 positive cells inparental lines vs. after 3 or 8 weeks treatment with erlotinib. (D)Quantification of integrin β3 (ITGβ3) gene expression in human lungcancer biopsies from patients from the BATTLE Study (18) who werepreviously treated with an EGFR inhibitor and progressed (n=27), versuspatients who were EGFR inhibitor naïve (n=39). (*P=0.04 using aStudent's t test). (E) Paired human lung cancer biopsies obtained beforeand after erlotinib resistance were immunohistochemically stained forintegrin β3. Scale bar, 50 (F) Right, effect of integrin β3 knockdown onerlotinib resistance of β3-positive cells. Cells were treated with 0.5μM of erlotinib. Results are normalized using non-treated cells ascontrols. n=3; mean±SEM. *P<0.05, **P<0.001. Left, effect of integrin β3ectopic expression on erlotinib resistance in FG and H441 cells. Cellswere treated with 0.5 μM of erlotinib. n=3; mean±SEM. *P<0.05,**P<0.001. (G) Right, effect of integrin β3 knockdown on erlotinibresistance in vivo, A549 shCTRL and A549 sh integrin β3 (n=8 pertreatment group) were treated with erlotinib (25 mg/kg/day) or vehicleduring 16 days. Results are expressed as average of tumor volume at day16. *P<0.05. Left, orthotopic FG and FG-β3 tumors (>1000 mm³; n=5 pertreatment group) were treated for 30 days with vehicle or erlotinib.Results are expressed as % tumor weight compared to vehicle control.*P<0.05.

FIG. 2 (FIG. 13/31) illustrates data showing that integrin β3 isrequired to promote KRAS dependency and KRAS-mediated EGFR inhibitorresistance.

(A) Confocal microscopy images show immunostaining for integrin β3(green), K-, N-, H-, R-Ras (red), and DNA (TOPRO-3, blue) for BxPc3cells grown in suspension in media with 10% serum. Arrows indicateclusters where integrin β3 and KRAS colocalize (yellow). Scale bar, 10μm. Data are representative of three independent experiments. ErlotinibIC₅₀ in a panel of human carcinoma cell lines expressing non-targetshRNA control or KRAS-specific shRNA and treated with erlotinib. n=3mean±SEM. *P<0.05, **P<0.01. (B-C) Confocal microscopy images showimmunostaining for integrin β3 (green), KRAs (red) and DNA (Topro-3,blue) for PANC-1 (KRAS mutant) and HCC827 (KRAS wild-type) afteracquired resistance to erlotinib (HCC827R) grown in suspension inabsence (Vehicle) or in presence of erlotinib (0.5 μM and 0.1 μMrespectively). Arrows indicate clusters where integrin β3 and KRAScolocalize (yellow). Scale bar, 10 μm. Data are representative of threeindependent experiments. (D) Effect of KRAS knockdown on tumorspheresformation in a panel of lung and pancreatic cancer cells expressing orlacking integrin β3. n=3 mean±SEM. *P<0.05, **P<0.01. (E) Effect of KRASknockdown on tumorsphere formation in PANC-1 (KRAS mutant) stablyexpressing non-target shRNA control (μ3-positive) or specific-integrinβ3 shRNA (β3 negative) in FG (KRAS mutant) and BxPc3 (KRAS wild-type)stably expressing vector control or integrin β3. *n=3; mean+SEM.*P<0.05. **P<0.01. (F) Effect of KRAS knockdown on erlotinib resistanceof β3-negative and β3-positive epithelial cancer cell lines. Cells weretreated with a dose response of erlotinib. n=3; mean±SEM, *P<0.05,**P<0.01. (G) Confocal microscopy images show immunostaining forintegrin β3 (green), KRAS (red) and DNA (TOPRO-3, blue) for PANC-1 cellsexpressing non-target shRNA control or Galectin 3-specific shRNA grownin suspension. Scale bar=10 μm. Data are representative of threeindependent experiments. (H) Top: immunoblot analysis of integrin β3immunoprecipitates from PANC-1 cells expressing non-target shRNA control(CTRL) or Galectin-3-specific shRNA (Gal-3). Bottom: immunoblot analysisof Galectin-3 immunoprecipitates from PANC-1 cells expressing non-targetshRNA control (CTRL) or integrin β3-specific shRNA (β3). Data arerepresentative of three independent experiments. (I) Erlotinib doseresponse of FG-β3 cells expressing a non-target shRNA control or aGalectin-3-specific shRNA (sh Gal-3). n=3; mean±SEM.

FIG. 3 (FIG. 14/31) illustrates data showing that RalB is a centralplayer of integrin β3-mediated EGFR inhibitor resistance.

(A) Effect of RalB knockdown on erlotinib resistance of β3-positiveepithelial cancer cell lines. Cells were treated with 0.5 μM oferlotinib. n=3; mean±SEM, *P<0.05, **P<0.01. (B) Effect of RalBknockdown on erlotinib resistance of β3-positive human pancreatic(FG-β3) orthotopic tumor xenografts. Established tumors expressingnon-target shRNA, (shCTRL) or a shRNA targeting RalB (sh RalB) (>1000mm³; n=13 per treatment group) were randomized and treated for 10 dayswith vehicle or erlotinib. Results are expressed as % of tumor weightchanges after erlotinib treatment compared to vehicle. **P<0.01. (C)Effect of expression of a constitutively active Ral G23V mutant onerlotinib response of β3 negative cells. Cells were treated with 0.5 μMof erlotinib. n=3; mean±SEM. *P<0.05. (D) Effect of expression ofintegrin β3 on KRAS and RalB membrane localization. Data arerepresentative of two independent experiments. (E) Ral activity wasdetermined in PANC-1 cells grown in suspension by using a GST-RalBP1-RBDimmunoprecipitation assay Immunoblots indicate RalB activity andassociation of active RalB with integrin β3. Data are representative ofthree independent experiments. (F) Confocal microscopy images ofintegrin αvβ3 (green), RalB (red) and DNA (TOPRO-3, blue) in tumorbiopsies from pancreatic cancer patients. Scale bar, 20 (G) Effect of β3expression and KRAS expression on RalB activity, measured using aGST-RalBP1-RBD immunoprecipitation assay. Data are representative ofthree independent experiments. (H) Immunoblot analysis of FG and FG-β3stably expressing non-target shRNA control or RalB-specific shRNA, grownin suspension and treated with erlotinib (0.5 μM). Data arerepresentative of three independent experiments. (I) Effect of TBK1 andp65 NFκB on erlotinib resistance of FG-β3 cells. Cells were treated with0.5 μM of erlotinib. n=3; mean±SEM. *P<0.05, **P<0.01.

FIG. 4 (FIG. 15/31) illustrates data showing that reversal ofβ3-mediated EGFR inhibitor resistance in oncogenic KRAS model bypharmacological inhibition.

(A) Effect of NFkB inhibitors on erlotinib response of β3-positive cells(FG-β3, PANC-1 and A549). Cells were treated with vehicle, erlotinib(0.5 lenalidomide (1-bortezomib (4 nM) alone or in combination. n=3;mean±SEM. *P<0.05, **P<0.01. (B) Left, mice bearing subcutaneousβ3-positive tumors (FG-β3) were treated with vehicle, erlotinib (25mg/kg/day), lenalidomide (25 mg/kg/day) or the combination of erlotiniband lenalidomide. Tumor dimensions are reported as the fold changerelative to size of the same tumor on Day 1. Mean±SEM, (A) *P=0.042using a one way ANOVA test. n=6 mice per group. Right, mice bearingsubcutaneous β3-positive tumors (FG-R) after acquired resistance toerlotinib were treated with vehicle, erlotinib (25 mg/kg/day),bortezomib (0.25 mg/kg), the combination of erlotinib and bortezomib.Tumor dimensions are reported as the fold change relative to size of thesame tumor on Day 1. *P=0.0134 using a one way ANOVA test. n=8 mice pergroup. (C) Model depicting the proposed integrin αvβ3-mediated KRASdependency and EGFR inhibitor resistance mechanism.

Supplementary FIG. S1 (FIG. 16/31) illustrates resistance to EGFRinhibitor is associated with integrin β3 expression in pancreatic andlung human carcinoma cell lines. (A) Immunoblots showing integrin β3expression in human cell lines used in FIG. 1A and FIG. 1B. (B) Effectof erlotinib on HCC827 xenograft tumors in immuno—compromised mice (n=5mice per treatment group) relative to vehicle-treated control tumors.Representative Integrin β3 cell surface quantification in HCC827 treatedwith vehicle or erlotinib during 64 days. (C) Integrin αvβ3quantification in orthotopic lung and pancreas tumors treated withvehicle or erlotinib until resistance. For lung cancer, integrin β3expression was scored (scale 0 to 3) and representative images areshown. For pancreatic cancer, integrin β3 expression was quantified asratio of integrin αvβ3 pixel area over nuclei pixel area usingMETAMORPH™ (**P=0.0012, *P=0.049 using Mann-Whitney U test).Representative immunofluorescent staining of integrin αvβ3 in pancreatichuman xenografts treated 4 weeks with vehicle or erlotinib.

Supplementary FIG. S2 (FIG. 17/31) illustrates Integrin β3 expressionpredicts intrinsic resistance to EGFR inhibitors in tumors. Plot ofprogression-free survival for erlotinib-treated patients with low vs.high protein expression of β3 integrin measured from non-small cell lungcancer biopsy material obtained at diagnosis (*P=0.0122, usingMann-Whitney U test). Representative images showing immunohistochemicalstaining for β3 integrin (brown) are shown.

Supplementary FIG. S3 (FIG. 18/31) illustrates Integrin β3 confersReceptor Tyrosine Kinase inhibitor resistance.

(A) Immunoblots showing integrin β3 knockdown efficiency in cells usedin FIG. 1. (B) Response of A549 lung carcinoma cells non-target shRNAcontrol or shRNA targeting integrin β3 to treatment with either vehicleor erlotinib (25 mg/kg/day) during 16 days. Tumor volumes are expressedas mean±SEM. n=8 mice per group. (C) Immunoblots showing expression ofindicated proteins of representative tumors. (D) Representativephotographs of crystal violet-stained tumorspheres of β3-negative andβ3-positive cells after erlotinib, OSI-906, gemcitabine and cisplatintreatment. (E) Effect of integrin β3 expression on lapatinib, OSI-906,cisplatin and gemcitabine n=3; mean±SEM. (F) Viability assay(CellTiter-Glo assay) of FG and FG-β3 cells grown in suspension in mediawith or without serum. n=2; mean+SEM. *P<0.05. **P<0.01.

Supplementary FIG. S4 (FIG. 19/31) illustrates Integrin β3-mediated EGFRinhibitor resistance is independent of its ligand binding.

Effect of ectopic expression of β3 wild-type (FG-β3) or the β3 D119A(FG-D119A) ligand binding domain mutant on erlotinib response. n=3;mean±SEM Immunoblot showing transfection efficiency of vector control,integrin β3 wild-type and integrin β3 D119A.Supplementary FIG. S5 (FIG. 20/31) illustrates Integrin β3 colocalizesand interacts with oncogenic and active wild-type KRAS.(A) Confocal microscopy images of FG and FG-β3 cells grown in suspensionin media 10% serum with or without erlotinib (0.5 μM) and stained forKRAS (red), integrin αvβ3 (green) and DNA (TOPRO-3, blue). Scale bar, 10μm. Data are representative of three independent experiments. (B) Rasactivity was determined in PANC-1 cells grown in suspension by using aGST-Rafl-RBD immunoprecipitation assay Immunoblots indicate KRASactivity and association of active KRAS with integrin β3. Data arerepresentative of three independent experiments. (C) Immunoblot analysisof Integrin αvβ3 immunoprecipitates from BxPC-3 cells grown insuspension in presence or absence of growth factors.

Supplementary FIG. S6 (FIG. 21/31) illustrates Integrin β3 expressionpromotes KRAS dependency.

(A) Immunoblots showing KRAS knockdown efficiency in cells used in FIG.2. (B) Representative photographs of crystal violet-stained tumorspheresof FG and A549 cells expressing non-target shRNA control orspecific-KRAS shRNA. (C) Effect of an additional KRAS knockdown ontumorspheres formation in PANC-1 stably expressing non-target shRNAcontrol (β3-positive) or specific-integrin β3 shRNA (β3 negative). n=3;mean+SEM. *P<0.05. Immunoblots showing KRAS knockdown efficiency.

Supplementary FIG. S7 (FIG. 22/31) illustrates KRAS and Galectin-3colocalize in integrin β3-positive cells.

Confocal microscopy images of FG and FG-β3 cells grown in suspension andstained for KRAS (green), galectin-3 (red) and DNA (TOPRO-3, blue).Scale bar, 10 μm. Data are representative of three independentexperiments.Supplementary FIG. S8 (FIG. 23/31) illustrates Integrin β3-mediated KRASdependency and erlotinib resistance is independent of ERK, AKT and RalA.(A) Effect of ERK, AKT, RalA and RalB knockdown on erlotinib response(erlotinib 0.5 μM) of β3-negative FG and in-positive FG-β3 cells.n=triplicate. (B) Immunoblots showing ERK, AKT RalA and RalB knockdownefficiency. (C) Immunoblots showing RalB knockdown efficiency in cellsused in FIG. 3.

Supplementary FIG. S9 (FIG. 24/31) illustrates Constitutive active NFkBis sufficient to promote erlotinib resistance.

(A) Immunoblots showing TBK1 and NFkB knockdown efficiency used in FIG.3. (B) Effect of constitutive active S276D p65NFkB on erlotinib response(erlotinib 0.5 μM) of β3-negative cells (FG cells). n=3; mean±SEM.*P<0.05.

Supplementary FIG. S10 (FIG. 25/31) illustrates NFkB inhibitors incombination with erlotinib increase cell death in vivo.

(A-B) Immunoblots showing expression of indicated proteins ofrepresentative tumors from shown in FIG. 4B (C) Confocal microscopyimages of cleaved caspase 3 (red) and DNA (TOPRO-3, blue) in tumorbiopsies from xenografts tumors used in FIG. 4B treated with vehicle,erlotinib, lenalidomide or lenalidomide and erlotinib in combo. Scalebar, 20 μm. (D) Confocal microscopy images of cleaved caspase 3 (red)and DNA (TOPRO-3, blue) in tumor biopsies from xenografts tumors used inFIG. 4B treated with vehicle, erlotinib, bortezomib or bortezomib anderlotinib in combo.

Supplementary Table 1: shows differentially expressed genes in cellsresistant to erlotinib (PANC-1, H1650, A459) compared with the averageof two sensitive cells (FG, H441) and in HCC827 after acquiredresistance in vivo (HCC827R) vs. the HCC827 vehicle-treated control. Thegenes upregulated more than 2.5 fold are in red.

Supplementary Table 2: shows KRAS mutational status of the pancreaticand lung cancer cell lines used in this study.

REFERENCES Example 2

-   1. R. J. Gillies, D. Verduzco, R. A. Gatenby, Evolutionary dynamics    of carcinogenesis and why targeted therapy does not work. Nature    reviews. Cancer 12, 487 (July, 2012).-   2. S. Zhang et al., Combating trastuzumab resistance by targeting    SRC, a common node downstream of multiple resistance pathways.    Nature medicine 17, 461 (April, 2011).-   3. J. S. Duncan et al., Dynamic reprogramming of the kinome in    response to targeted MEK inhibition in triple-negative breast    cancer. Cell 149, 307 (Apr. 13, 2012).-   4. D. L. Wheeler, E. F. Dunn, P. M. Harari, Understanding resistance    to EGFR inhibitors-impact on future treatment strategies. Nature    reviews 7, 493 (September, 2010).-   5. F. Ciardiello, G. Tortora, EGFR antagonists in cancer treatment.    The New England journal of medicine 358, 1160 (Mar. 13, 2008).-   6. C. M. Ardito et al., EGF receptor is required for KRAS-induced    pancreatic tumorigenesis. Cancer Cell 22, 304 (Sep. 11, 2012).-   7. C. Navas et al., EGF receptor signaling is essential for k-ras    oncogene-driven pancreatic ductal adenocarcinoma. Cancer Cell 22,    318 (Sep. 11, 2012).-   8. C. Ferte et al., Durable responses to Erlotinib despite KRAS    mutations in two patients with metastatic lung adenocarcinoma. Ann    Oncol 21, 1385 (June, 2010).-   9. M. J. Moore et al., Erlotinib plus gemcitabine compared with    gemcitabine alone in patients with advanced pancreatic cancer: a    phase III trial of the National Cancer Institute of Canada Clinical    Trials Group. J Clin Oncol 25, 1960 (May 20, 2007).-   10. E. S. Kim et al., The BATTLE Trial: Personalizing Therapy for    Lung Cancer. Cancer discovery 1, 44 (June, 2012).-   11. J. S. Desgrosellier et al., An integrin alpha(v)beta(3)-c-Src    oncogenic unit promotes anchorage-independence and tumor    progression. Nature medicine 15, 1163 (October, 2009).-   12. A. U. Newlaczyl, L. G. Yu, Galectin-3—a jack-of-all-trades in    cancer. Cancer letters 313, 123 (Dec. 27, 2011).-   13. A. I. Markowska, F. T. Liu, N. Panjwani, Galectin-3 is an    important mediator of VEGF- and bFGF-mediated angiogenic response.    The Journal of experimental medicine 207, 1981 (Aug. 30, 2010).-   14. D. A. Barbie et al., Systematic RNA interference reveals that    oncogenic KRAS-driven cancers require TBK1. Nature 462, 108 (Nov. 5,    2009).-   15. Y. Chien et al., RalB GTPase-mediated activation of the IkappaB    family kinase TBK1 couples innate immune signaling to tumor cell    survival. Cell 127, 157 (Oct. 6, 2006).-   16. Y. Yang et al., Exploiting Synthetic Lethality for the Therapy    of ABC Diffuse Large B Cell Lymphoma. Cancer Cell 21, 723 (Jun. 12,    2012).-   17. M. S. Kumar et al., The GATA2 transcriptional network is    requisite for RAS oncogene-driven non-small cell lung cancer. Cell    149, 642 (Apr. 27, 2012).-   18. E. S. Kim et al., The BATTLE Trial: Personalizing Therapy for    Lung Cancer. Cancer discovery, (Apr. 3, 2011, 2011).

Example 3 A β3 Integrin/KRAS Complex Shift Tumor Phenotype TowardStemness

The data presented herein demonstrates the effectiveness of thecompositions and methods of the invention in reversing tumor initiationand self-renewal, and resensitizing tumors to Receptor Tyrosine Kinase(RTK) inhibition.

Integrin αvβ3 expression is a marker of tumor progression for a widerange of histologically distinct cancers¹, yet the molecular mechanismby which αvβ3 influences the growth and malignancy of cancer is poorlyunderstood. Here, we reveal that integrin αvβ3, in the unligated state,is both necessary and sufficient to promote tumor initiation andself-renewal through its recruitment of KRAS/RalB to the plasma membraneleading to the activation of TBK-1/NFkB. Accordingly, this pathway alsodrives KRAS-mediated resistance to receptor tyrosine kinases inhibitorssuch as erlotinib Inhibition of RalB or its effectors not only reversestumor initiation and self renewal but resensitizes tumors to ReceptorTyrosine Kinase (RTK) inhibition. These findings provide a molecularbasis to explain how αvβ3 drives tumor progression and reveals atherapeutic strategy to target and destroy these cells.

Tumor-initiating cells (also known as cancer stem cells), EMT, and drugresistance have recently been linked together as a challenge for cancertherapy². Here, we propose integrin αvβ3 as a potential lynchpin capableof influencing and integrating these three critical determinants ofcancer progression. Indeed, expression of β3 integrin has long beenassociated with poor outcome and higher incidence of metastasis for avariety of epithelial cancers¹, its expression has been reported on asubpopulation of breast^(3,4) and myeloid leukemia cancer stem cells,and β3 has been implicated in the process of epithelial-to-mesenchymaltransition, especially in the context of TGF-β^(5,6). Although theprimary influence of integrins is considered to be their regulation ofcell-matrix adhesion events leading to clustering of focal adhesions todrive intracellular signaling cascades, we have recently made thesurprising observation that αvβ3 integrin is capable of forming clusterson the surface of non-adherent cells to recruit signaling complexes thatcan drive cell survival in the absence of ligand binding⁷. This propertyis not shared by other integrins, including β1, suggesting that αvβ3expression may provide a critical survival signal for cells invadinghostile environments. Indeed, exposing quiescent endothelial cells toangiogenic growth factors results in the upregulation of αvβ3 expressionthat is required for their conversion to the angiogenic/invasive state⁸.We propose that expression of αvβ3 offers tumor cells an equivalentsurvival advantage, and that targeting this pathway could undercut atumors ability to metastasize and resist therapy.

Since we previously reported that integrin αvβ3 expression wasassociated with increased anchorage-independent growth⁷, we postulatedthat β3 expression may play a role in tumor progression by shiftingepithelial tumor cells toward a stem-like phenotype. To evaluate apossible effect of β3 expression on tumor stemness in vivo, we knockeddown integrin β3 in various human carcinoma cells expressing thisreceptor, or ectopically expressed β3 in tumor cells lacking thisintegrin. Compared with their respective β3-negative counterparts,β3-positive cells showed a 50-fold increased tumor-initiating capacity,measured as a higher frequency of tumor initiating cells in a limitingdilution assay (see FIG. 1 a and FIG. S1 a-c (of Example 3), which areFIG. 32 a and FIGS. 36 a, 36 b and 36 c, respectfully).

In vitro, tumor stemness is also associated with an increased capacityto form tumorspheres and undergo self-renewal. Consequently, we measuredthe capacity of β3 expressing tumor cells to form primary and secondarytumorspheres. Notably, the ratio of secondary tumorspheres to primarytumorspheres was 2-4 fold higher for cells expressing integrin β3 (seeFIG. 1 b-d and FIG. S1 c (of Example 3); which are FIG. 32 b-d and FIG.36 c, respectively). Together, these findings indicate that β3expression enhances the stem-like behavior of these tumors.

Tumor-initiating cells are known to be particularly resistant tocellular stresses, such as nutrient deprivation or exposure toanti-cancer drugs⁹. Indeed, β3-positive cells survived to a greaterdegree when stressed by removal of serum from their growth mediacompared with cells lacking this integrin (FIG. S1 d (of Example 3), orFIG. 36 d). However, β3 expression did not impact the response to thechemotherapeutic agent cisplatin or the anti-metabolite agentgemcitabine for cells growing in 3D (FIG. 2 a, or FIG. 33 a). Underthese same conditions, β3 expression did strongly correlate with reducedsensitivity to Receptor Tyrosine Kinase (RTK) inhibitors, including theEGFR1 inhibitor erlotinib, the EGFR1/EGFR2 inhibitor lapatinib, and theIGF-1R inhibitor linsitinib (OSI906) (FIG. 2 b-c, or FIG. 33 b-c).

This link between β3 expression and RTK inhibitor resistance was alsoobserved in vivo, as knockdown of integrin β3 overcame erlotinibresistance for subcutaneous A549 xenografts (FIG. 2 d, or FIG. 33 d),while ectopic expression of integrin β3 conferred erlotinib resistanceto FG tumors growing orthotopically in the pancreas (FIG. 2 e, or FIG.33 e).

In clinic, human non-small cell lung cancer harboring activatingmutations in EGFR often initially respond to erlotinib but invariablydevelop resistance through multiple mechanisms including acquired orselected mutations, gene amplification and alternate routes of kinasepathway activation. Recent studies indicate that multiple resistancemechanisms may operate within an individual tumor to promote acquiredresistance to EGFR TKIs in persons with NSCLC and accumulating evidencesupports the concept that the tumor-initiating cells contribute to EGFRTKI resistance in lung. To assess the clinical relevance of ourfindings, mice with established HCC827 (human NSCLC cells with deletionof exon 19 of EGFR) have been treated with erlotinib until developmentof acquired resistance (FIG. 2 f, or FIG. 33 f). Integrin β3 expressionwas significantly higher in erlotinib resistant tumors compared tovehicle-treated tumors (FIG. 2 g, or FIG. 33 g).

To validate these findings, we examined biopsies from lung cancerpatients harboring an EGFR mutation before erlotinib treatment and afteracquired resistance and we found that integrin β3 expression wasqualitatively higher after acquired resistance to erlotinib (FIG. 2 h,or FIG. 33 h; FIG. S1 e, or, or FIG. 36 e). To investigate the role ofintegrin β3 in this context, we sorted erlotinib-resistant HCC827 tumorsinto integrin β3⁺ and Integrin β3⁻ populations and tested them for tumorinitiating cell abilities. As expected, the integrin β3⁺ populationshowed enhanced tumor initiating and self-renewal capacities compared tothe integrin β3⁻ population (FIG. 2 i j, or FIG. 33 i-j; FIG. S1 f, orFIG. 36 f) suggesting that integrin β3 contribute to the stem-likephenotype of the drug resistance tumor. In addition integrin β3 has beenfound in a subpopulation of the CD166+ cells in human adenocarcinomaafter acquired resistance to erlotinib (FIG. S1 g, or FIG. 36 g).Together these findings reveal that β3 expression is both necessary andsufficient to account for tumor stem-like properties in vitro and invivo.

Our results suggest that targeting integrin β3 function may represent aviable approach to reverse stem-like properties and sensitize tumors toRTK inhibitors. However, integrin antagonists that compete for ligandbinding sites and disrupt cell adhesion are not likely to have an impacton the stemness and drug resistance properties that are represented by3D growth of tumor cells under anchorage-independent conditions.Accordingly, neither expression of a mutant integrin β3 (D119A)incapable of binding ligand nor treating cells with cyclic peptides thatcompete with αvβ3 for ligand binding impacted the β3-mediatedenhancement of 3D colony formation in the presence of erlotinib (FIG. S2a-b, or FIG. 37 a-b). Thus, the contribution of β3 integrin to stemnessand drug resistance appears to involve a non-canonical function for thisintegrin, independent from its traditional role as a mediator of celladhesion to specific β3 ligands. If this is the case, then blocking thispathway will require understanding the downstream molecular mechanism(s)that become engaged in the presence of β3.

To study how β3 integrin influences tumor stemness, we considered thatintegrins frequently transmit signals in the context of RAS familymembers¹⁰. To examine a possible link between β3 expression and RAS,tumor cells growing in 3D were stained for β3 and various RAS familymembers. Interestingly, in cells growing in suspension, β3 co-localizedin clusters at the plasma membrane with KRAS, but not with NRAS, RRAS,or HRAS (FIG. 3 a, or FIG. 34 a, FIG. S2 c, or FIG. 37 c). In fact, KRAScould be specifically co-immunoprecipitated with β3 but not 31 integrin(FIG. 3 b, or FIG. 34 b), indicating a specific interaction between β3and KRAS in cells undergoing anchorage-independent growth. Finally, weobserved that KRAS knockdown abolished the β3-induced anchorageindependence, self-renewal, and erlotinib resistance (FIG. 3 c-e, orFIG. 34 c-e), indicating that β3 and KRAS cooperate to drive β3-mediatedstem-like phenotype.

Since there are no known KRAS binding sites on the β3 cytoplasmic tail,it is likely that this KRAS/β3 interaction occurs through anintermediary. Galectin-3 is a carbohydrate-binding lectin linked totumor progression¹¹ that is known to separately interact with KRAS¹² andintegrin αvβ3¹³. Therefore, we considered whether Galectin-3 might serveas an adaptor facilitating the β3/KRAS interaction inanchorage-independent tumor cells. Indeed, we observed co-localizationof β3, KRAS, and Galectin-3 within membrane clusters for cells grownunder anchorage-independent conditions (FIG. 3 f, or FIG. 34 f).Knockdown of Galectin-3 not only prevented formation of the KRAS/β3complex (FIG. 3 f-g, or FIG. 34 f-g), but also reversed the advantage ofβ3 expression for anchorage independence erlotinib resistance andself-renewal (FIG. 3 h-i, or FIG. 34 h-i). These findings provideevidence that Galectin-3 facilitates an interaction between β3 and KRASthat is required for the promotion of stemness.

The activation of KRAS elicits changes in cellular function by signalingthrough a number of downstream effectors, most prominently AKT/PI3K,RAF/MEK/ERK, and Ral GTPases¹⁴. Depletion of Akt, Erk, or RalA inhibitedthe 3D growth of β3⁺ versus β3⁻ tumor cells equally (FIG. S3 a-b, orFIG. 38 a-b), suggesting these effectors were not selectively involvedin the ability of β3 to enhance stemness. In contrast, knockdown of RalBnot only selectively impaired colony formation for β3⁺ cells (FIG. 4 a,or FIG. 35 a; FIG. S3 c-d), but it also negated the effect of β3expression and stem-like phenotype (FIG. 4 b-c; FIG. S3 e, or FIG. 38 e)and erlotinib resistance (FIG. 4 d-e, or FIG. 35 d-e). Mechanistically,the association between KRAS and integrin β3 at the plasma membrane wasable to recruit and activate RalB (Supplementary Information, FIG. S3f-h, or FIG. 38 f-h). In fact, the activation of RalB alone issufficient to drive this pathway, since expression of a constitutivelyactive RalB G23V mutant in β3-negative tumor cells conferred erlotinibresistance (FIG. S3 i, or FIG. 38 i).

Consistent with recent studies that have linked the RalB effectors TBK1and RelA to RTKI resistance and stemness¹⁵, β3⁺ tumor cells showedactivation of these effectors even in the presence of erlotinib (FIG. 4f, or FIG. 35 f). Loss of RalB restored erlotinib-mediated inhibition ofTBK1 and RelA for β3⁺ tumor cells (FIG. 4 f, or FIG. 35 f), suggestingthese as therapeutic targets relevant for this pathway. Since targetingintegrin ligation events cannot perturb this pathway, and RAS inhibitorshave underperformed expectations in the clinic, interrupting signalingdownstream of RalB could reverse the stemness potential of β3⁺ tumorcells. Indeed, genetic or pharmacological inhibition of TBK1 or RelAovercame self-renewal and β3-mediated erlotinib resistance (FIG. 4 g-i,or FIG. 35 g-i; FIG. S4 a-e, or FIG. 39 a-e). Taken together, ourobservations indicate that integrin β3 expression promotes a cancerstem-like program by cooperating with KRAS to regulate the activity ofRalB, and that elements of this pathway can be disrupted to providetherapeutic benefit in mouse models of lung and pancreatic cancer.

Despite numerous advances in our knowledge of cancer, most advancedcancers remain incurable. At present, conventional therapies can controltumor growth initially but most patients ultimately relapse,highlighting the urgent need for new approaches to treat canceroustumors. One such approach may be to target the tumor-initiating cells.An emerging picture is that tumor-initiating cells do not constitute ahomogenous population of cells explaining the lack of reliability ofcancer stem markers. We discovered an integrin β3+ subpopulation oftumor-initiating cells that are specifically resistant to RTKIs. Severalstudies have shown that integrin-mediated cellular adhesion toextracellular matrix components is an important determinant oftherapeutic response. In fact, integrin β3 increases adhesion-mediatedcell survival, drug resistance and suppresses antitumor immunity¹⁶suggesting that blocking integrin β3 could offer a therapeutic strategy.We and other previously established that besides the adhesion-dependentfunctions, integrins can also be involved in different cellularmechanisms. In fact, we recently showed the ability of β3 to driveanchorage-independent growth in 3D without providing any growth orsurvival advantage in 2D⁷. Since there is also evidence that 3D culturesmimic drug sensitivity in vivo more accurately than 2D cultures¹⁷, wefocused on the role of β3 in promoting stemness and drug resistanceusing 3D culture models in vitro and tumor growth in vivo.

Although KRAS mutations, present in 95% of pancreatic tumors and 25% oflung cancers, have been linked to RTK inhibitor resistance, recentstudies have demonstrated that expression of oncogenic KRAS is anincomplete predictor of erlotinib resistance in pancreatic and lungcancer, since a number of individual patients presenting with KRASmutation unexpectedly respond to therapy. In fact, for 3D growth in softagar and in vivo experiments, we found that erlotinib resistance couldbe predicted by evaluating integrin β3 expression in KRAS mutant cancerssuggesting that oncogenic KRAS is not sufficient to drive erlotinibresistance. It has been demonstrated that its localization to the plasmamembrane is a critical component to its function and inhibiting itsmembrane localization could represent a therapeutic strategy. Here, werevealed an unexpected role for integrin b3 that can maintain KRAS inmembrane clusters through its interaction with Galectin-3 representing apotential therapeutic opportunity. KRAS dependency had previously beenlinked to erlotinib sensitivity for tumor cells growing in 2D¹⁸. Theseresults emphasize the contribution of β3 integrin to tumor cell behaviorfor cells grown in 3D, and suggest that alternative or even opposingpathways may dominate when cells are grown in 2D under adherentconditions.

The invention thus provides methods for determining or predicting thecourse of cancer therapy in terms of personalized medicine. Our resultsdemonstrate that biopsies taken at diagnosis can be screened for β3expression to predict a poor response to RTK-targeted therapies. If abiopsy is positive, we would predict that co-administering an inhibitorof RalB/TBK1/RelA could improve the response. Since β3⁺ tumor cells areparticularly sensitive to KRAS knockdown, such tumors represent apopulation of particularly good candidates for KRAS-directed therapieswhich have shown only poor responses thus far.

Our work demonstrates that a tumor could be sensitized to therapy byreversing the advantages of β3 expression. We demonstrate this can beachieved by inhibiting RalB-mediated signaling using genetic knockdownor by treating with a number of FDA-approved drugs. We focused ourefforts on the role of β3 expression on lung and pancreatic cancers inthe context of erlotinib therapy, since it is approved for thesepatients. However, we were able to correlate KRAS dependency and β3expression for a diverse panel of epithelial cancer cells.

Methods

Compounds and Cell Culture.

Human pancreatic (FG, PANC-1), breast (MDAMB231 (MDA231) and lung (A549and H1650) cancer cell lines were grown in ATCC recommended mediasupplemented with 10% fetal bovine serum, glutamine and non-essentialamino acids. We obtained FG-β3, FG-D119A mutant and PANC-shβ3 cells aspreviously described. Erlotinib, linsitinib, Gemcitabine, Bortezomib andLapatinib were purchased from Chemietek. Cisplatin was generated fromSigma-Aldrich.

Self Renewal Tumorsphere Assay and Soft Agar Assay.

Tumorsphere assay was performed as previously described. Soft agarformation assays were performed essentially as described previously.Cells were treated with vehicle (DMSO), erlotinib (10 nM to 5 μM),lapatinib (10 nM to 5 μM), gemcitabine (0.001 nM to 5 μM), linsitinib(10 nM to 5 μM), cisplatin (10 nM to 5 μM), or bortezomib (4 nM) dilutedin DMSO. The media was replaced with fresh inhibitor 2/5 times a week.Survival curves were generated at least with five concentration points.

Limiting Dilution.

All mouse experiments were carried out in accordance with approvedprotocols from the UCSD animal subjects committee and with theguidelines set forth in the NIH Guide for the Care and Use of LaboratoryAnimals. 10², 10³, 10⁴, 10⁵ and 10⁶ of A549 NS, A549 shβ3, FG, FG-β3 andFG-β3 sh RalB cells were suspended in a mixture of Basement MembraneMatrix Phenol Red-free (BD Biosciences) and PBS 1:1 and injected in theflanks of 6/8 weeks old female immune compromised nu/nu mice. After30/40 days, palpable tumors were counted and the tumor-initiating cellsfrequency was calculated using the ELDA software.

Orthotopic Pancreas Cancer Xenograft Model.

Tumors were generated as previously described (JAY). Tumors wereestablished for 2-3 weeks (tumor sizes were monitored by ultrasound)before beginning dosing. Mice were dosed by oral gavage with vehicle (6%captisol) or 10, 25 and 50 mg/kg/day erlotinib for 10 to 30 days priorto harvest.

Immunofluorescence Microscopy.

Frozen sections from tumors from patients diagnosed with pancreas ortumor cell lines were processed as previously described (Mielgo). Cellswere stained with indicated primary, followed by secondary antibodiesspecific for mouse or rabbit (Invitrogen), as appropriate. Samplesimaged on a Nikon Eclipse C1 confocal microscope with 1.4 NA 60×oil-immersion lens, using minimum pinhole (30 μm). Colocalizationbetween Integrin β3 and KRAS was studied using the Zenon AntibodyLabeling Kits (Invitrogen) and the KRAS rabbit antibody.

Biopsies from NSCLC Patients.

Tumor biopsies from University of California, San Diego (UCSD) MedicalCenter breast, pancreas and non-small cell lung cancer patients wereobtained. This work was approved by the UCSD Institutional Review Board(IRB).

Cell Viability Assay.

Cell viability assays were performed as described¹². Briefly cells wereseeded in low adherent plates 7 days in DMEM containing 10% or 0% serum,0.1% BSA.

Genetic knockdown and expression of mutant constructs.

Cells were transfected with vector control, WT, G23V RalB-FLAG, using alentiviral system. For knock-down experiments, cells were transfectedwith KRAS, RalA, RalB, AKT1, ERK1/2, TBK1, siRNA (Qiagen) using thelipofectamine reagent (Invitrogen) following manufacturer's protocol ortransfected with shRNA (Open Biosystems) using a lentiviral system. Genesilencing was confirmed by immunoblots analysis.

Immunohistochemical Analysis.

Immunostaining was performed according to the manufacturer'srecommendations (Vector Labs) on 5 M sections of paraffin-embeddedtumors from tumor biopsies from lung cancer patients. Tumor sectionswere processed as previously described²⁷ using integrin β3 (Abcam)+stemmarkers, diluted 1:200. Sections stained with integrin β3 were scored bya H-score according to the staining intensity (SI) on a scale 0 to 3within the whole tissue section.

RNA Extraction PCR

Immunoprecipitation and Immunoblots.

Lysates from cell lines and xenograft tumors were generated usingstandard methods and RIPA or Triton buffers. Immunoprecipitationexperiments were performed as previously described⁵⁹ withanti-integrin-3 (LM609) or Galectin-3. For immunoblot analysis, 25 μg ofprotein was boiled in Laemmli buffer and resolved on 8% to 15% gel. Thefollowing antibodies were used: anti-integrin β3 ( ), KRAS, NRAS, RRAS,HRAS, FAK and Hsp90 from Santa Cruz, phospho-S172 NAK/TBK1 fromEpitomics, TBK1, phospho-p65NFκB S276, p65NFκB, RalB, phospho-EGFR,EGFR, phospho-FAK Tyr 861 from Cell Signaling Technology, and Galectin 3from BioLegend.

Affinity Pull-Down Assays for Ras and Ral.

RAS and Ral activation assays were performed in accordance with themanufacturer's (Upstate) instruction. Briefly, cells were cultured insuspension for 3 h. 10 μg of Ral Assay Reagent (Ral BP1, agarose) or RASassay reagent (Raf-1 RBD, agarose) was added to 500 mg to 1 mg of totalcell protein in MLB buffer (Millipore). After 30 min of rocking at 4°C., the activated (GTP) forms of RAS/Ral bound to the agarose beads werecollected by centrifugation, washed, boiled in Laemmli buffer, andloaded on a 15% SDS-PAGE gel.

Statistical Analyses.

All statistical analyses were performed using Prism software (GraphPad).Two-tailed Mann Whitney U tests, Chi-squared tests, Fisher's exacttests, one way ANOVA tests or t-tests were used to calculate statisticalsignificance. A P value<0.05 was considered to be significant.

FIGURE LEGENDS Example 3

FIG. 1: Integrin β3 expression increase tumor-initiating andself-renewal capacities:

(a) Limiting dilution in vivo determining the frequency oftumor-initiating cells for A549 cells expressing non-target shRNAcontrol or integrin β3-specific shRNA and for FG cells expressingcontrol vector or integrin β3 (FG-β3). The frequency of tumor-initiatingcells per 10,000 cells was calculated using the ELDA extreme limitingdilution software. (b-c-d) Self-renewal capacity of A549 and PANC-1cells expressing non-target shRNA control (CTRL) or integrin β3-specificshRNA and of FG expressing control vector or integrin β3 (FG-β3),measured by quantifying the number of primary and secondarytumorspheres. Representative images of tumorspheres are shown. n=3;mean±SEM. *P<0.05, **P<0.01.

FIG. 2: Integrin β3 drives resistance to EGFR inhibitors:

(a) Effect of integrin β3 expression (ectopic expression for FG andintegrin β3-specific knockdown for PANC-1) cells on drug treatmentresponse. Cells were treated with a dose response of gemcitabine,cisplatin, erlotinib, lapatinib and linsitinib. Results are normalizedusing non-treated cells as controls. n=3; mean±SEM. *P<0.05, **P<0.001.(b) Effect of integrin β3 knockdown on erlotinib response in MDA-MB-231(MDA231), A549 and H1650. n=3; mean±SEM. *P<0.05, **P<0.001. (c) Effectof integrin β3 knockdown on erlotinib resistance in vivo, A549 shCTRLand A549 shβ3 (n=8 per treatment group) were treated with erlotinib (25mg/kg/day) or vehicle during 16 days. Tumor volumes are expressed asmean±SEM. *P<0.05. (d) Orthotopic FG and FG-β3 tumors (>1000 mm³; n=5per treatment group) were treated for 30 days with vehicle or erlotinib.Results are expressed as % tumor weight compared to vehicle control.*P<0.05. (e) Effect of erlotinib treatment on HCC827 xenograft tumors(n=8 tumors per treatment group). HCC827 cells were treated with vehiclecontrol or erlotinib (12.5 mg/kg/day) until acquired resistance. (f)Relative mRNA expression of integrin β3 (ITGB3) in HCC827vehicle-treated tumors (n=5) or erlotinib-treated tumors (n=7) from (e)after acquired resistance. Data are mean±SE; **P<0.001. (g) H&E sectionsand immunohistochemical analysis of integrin β3 expression in pairedhuman lung cancer biopsies obtained before and after erlotinibresistance. Scale bar, 50 μm. (h) Limiting dilution in vivo determiningthe frequency of tumor-initiating cells for HCC827 vehicle-treated(vehicle) and erlotinib-treated tumors from (erlotinib resistantnon-sorted) (e). The HCC827 erlotinib-treated tumors have been digestedand sorted in two groups: the integrin β3− and the integrin β3+population. (i) and (j) Self-renewal capacity of HCC827 vehicle-treated(vehicle), erlotinib-treated (erlotinib resistant non-sorted),erlotinib-treated integrin β3− population and erlotinib-treated integrinβ3+ population, measured by quantifying the number of primary andsecondary tumorspheres. n=3; mean±SEM. *P<0.05, **P<0.01.

FIG. 3: Integrin β3/KRAS complex is critical for integrin β3-mediatedstemness:

(a) Confocal microscopy images show immunostaining for Integrin β3(green), KRAS (red) and DNA (TOPRO-3, blue) for FG-β3, PANC-1, A549 andHCC827 after acquired resistance to erlotinib (HCC827 ER) grown insuspension. Arrows indicate clusters where integrin β3 and KRAScolocalize (yellow). Scale bar=10 μm. Data are representative of threeindependent experiments. (b) Ras activity was determined in PANC-1 cellsgrown in suspension by using a GST-Rafl-RBD immunoprecipitation assayImmunoblots indicate KRAS activity and association of active KRAS withintegrin β3. Data are representative of three independent experiments.(c) Effect of KRAS knockdown on tumorspheres formation in lung (A549 andH441) and pancreatic (FG and PANC-1) cancer cells expressing or lackingintegrin β3. n=3 mean±SEM. *P<0.05, **P<0.01. (d) Effect of KRASknockdown on erlotinib resistance of β3-negative and β3-positiveepithelial cancer cell lines. Cells were treated with a dose response oferlotinib. n=3; mean±SEM, *P<0.05, **P<0.01. (e) Self-renewal capacityof FG-β3 cells expressing non-target shRNA control (shCTRL) orKRAS-specific shRNA measured by quantifying the number of primary andsecondary tumorspheres. n=3; mean±SEM. *P<0.05, **P<0.01. (f) Confocalmicroscopy images show immunostaining for integrin β3 (green), KRAS(red) and DNA (TOPRO-3, blue) for PANC-1 cells expressing non-targetshRNA control or Galectin 3-specific shRNA grown in suspension. Scalebar=10 μm. Data are representative of three independent experiments. (g)immunoblot analysis of integrin β3 immunoprecipitates from PANC-1 cellsexpressing non-target shRNA control (CTRL) or Galectin-3-specific shRNA(Gal-3). Data are representative of three independent experiments. (h)Effect of Galectin-3 knockdown on integrin β3-mediated anchorageindependent growth and erlotinib resistance. PANC-1 cells expressing anon-target shRNA control or a Galectin-3-specific shRNA (sh Gal-3) weretreated with vehicle or erlotinib (0.5 μM). n=3; mean±SEM. (i)Self-renewal capacity of PANC-1 cells expressing non-target shRNAcontrol (shCTRL) or Galectin-3-specific shRNA (sh Gal-3) measured byquantifying the number of primary and secondary tumorspheres. n=3;mean±SEM. *P<0.05, **P<0.01.

FIG. 4. RalB/TBK1 signaling is a key modulator of integrin β3-mediatedstemness:

(a) Effect of RalB knockdown on anchorage independence. n=3; mean±SEM,*P<0.05, **P<0.01. (b) Self-renewal capacity of FG-β3 cells expressingnon-target shRNA control (sh CTRL) or RalB-specific shRNA (sh RalB)measured by quantifying the number of primary and secondarytumorspheres. n=3; mean±SEM. *P<0.05, **P<0.01. (c) Limiting dilution invivo determining the frequency of tumor-initiating cells for FG-β3 cellsexpressing non-target shRNA control or integrin RalB-specific shRNA. (d)Effect of RalB knockdown on erlotinib resistance of β3-positiveepithelial cancer cell lines. Cells were treated with 0.5 μM oferlotinib. n=3; mean±SEM, *P<0.05, **P<0.01. (e) Effect of RalBknockdown on erlotinib resistance of β3-positive human pancreatic(FG-β3) orthotopic tumor xenografts. Established tumors expressingnon-target shRNA, (sh CTRL) or a shRNA targeting RalB (sh RalB) (>1000mm³; n=13 per treatment group) were randomized and treated for 10 dayswith vehicle or erlotinib. Results are expressed as % of tumor weightchanges after erlotinib treatment compared to vehicle. *P<0.05. (f)Immunoblot analysis of FG and FG-β3 stably expressing non-target shRNAcontrol or RalB-specific shRNA, grown in 3D and treated with erlotinib(0.5 μM). Data are representative of three independent experiments. (g)Effect of TBK1 knockdown on PANC-1 self-renewal capacity. n=3; mean±SEM.*P<0.05, **P<0.01. (h) Effect of TBK1 knockdown on erlotinib resistanceof PANC-1 cells. Cells were treated with 0.5 μM of erlotinib. n=3;mean±SEM. *P<0.05, **P<0.01. (i) Mice bearing subcutaneous β3-positivetumors (PANC-1) were treated with vehicle, erlotinib (25 mg/kg/day),amlexanox (25 mg/kg/day) or the combination of erlotinib and amlexanox.Tumor dimensions are reported as the fold change relative to size of thesame tumor on Day 1. Mean±SEM, (A) *P=0.042 using a one way ANOVA test.n=8 mice per group.

FIG. S1—Example 3

(a-b) Limiting dilution tables. (c) Immunoblots showing integrin β3knockdown or ectopic expression efficiency in cells used in FIG. 1. (d)Viability assay (CellTiter-Glo assay) of FG and FG-β3 cells grown in 3Din media with or without serum. n=3; mean+SEM. *P<0.05. **P<0.01. (e)Immunohistochemical analysis of integrin β3 expression in paired humanlung cancer biopsies obtained before and after erlotinib resistance.Scale bar, 50 μm. (f) Limiting dilution table. (g) Immunohistochemistrystaining of CD166 and integrin β3 in human lung tumor biopsies afterEGFR™ acquired resistance.

FIG. S2—Example 3

(a) Effect of cilengetide treatment on erlotinib resistance in FG-β3 andPANC-1 cells. n=3; mean+SEM. (b) Effect of ectopic expression of β3wild-type (FG-β3) or the β3 D119A (FG-D119A) ligand binding domainmutant on erlotinib response. n=3; mean±SEM Immunoblot showingtransfection efficiency of vector control, integrin β3 wild-type andintegrin 3 D119A. (c) Confocal microscopy images of FG-β3 cells grown in3D and stained for integrin—β3 (green) and RAS family members (red).Scale bar, 10 μm. Data are representative of three independentexperiments. (d) Immunoblots showing KRAS knockdown efficiency in cellsused in FIG. 3. (e) Representative photographs of crystal violet-stainedtumorspheres of FG and A549 cells expressing non-target shRNA control orspecific-KRAS. (f) Effect of a second KRAS knockdown (shKRAS 2) ontumorspheres formation in PANC-1 stably expressing non-target shRNAcontrol (3-positive) or specific-integrin-β3 shRNA (3 negative). n=3;mean+SEM. *P<0.05.

FIG. S3—Example 3

(a) Effect of ERK, AKT and RalA knockdown on erlotinib response ofβ3-negative FG and 3-positive FG-3 cells. (b) Immunoblots showing ERK,AKT and RalA knockdown efficiency in cells used in (a). (c) Immunoblotsshowing RalB knockdown efficiency in cells used in FIG. 3. (d) Effect ofa second RalB knockdown (shRalB 2) on tumorspheres formation in PANC-1stably expressing non-target shRNA control (β3-positive) orspecific-integrin β3 shRNA (β3 negative). n=3; mean+SEM. *P<0.05. (e)Limiting dilution table. (f) Confocal microscopy images of integrin αvβ3(green), RalB (red) and DNA (TOPRO-3, blue) in tumor biopsies frompancreatic cancer patients. Scale bar, 20 μm. (g) Ral activity wasdetermined in PANC-1 cells grown in suspension by using a GST-RalBP1-RBDimmunoprecipitation assay Immunoblots indicate RalA and RalB activities.Data are representative of three independent experiments. (h) Effect ofβ3 expression and KRAS expression on RalB activity, measured using aGST-RalBP1-RBD immunoprecipitation assay. Data are representative ofthree independent experiments. (i) Effect of expression of aconstitutively active Ral G23V mutant on erlotinib resistance of β3positive and negative cells. n=3; mean±SEM. *P<0.05.

FIG. S4—Example 3

(a) Immunoblot showing TBK1 knockdown efficiency in PANC-1 cells used inFIG. 4. (b) Effect of theTBKl inhibitor amlexanox on erlotinib responseof PANC-1 cells. Cells were treated with vehicle, erlotinib (0.5 μM),amlexanox alone or in combination. (c) Effect of the NFkB inhibitorborthezomib on β3-positive cells (FG-β3, PANC-1 and A549). Cells weretreated with vehicle, erlotinib (0.5 μM), bortezomib (4 nM) alone or incombination. n=3; mean±SEM. *P<0.05, **P<0.01. (d) Mice bearingsubcutaneous β3-positive tumors (FG-β3) were treated with vehicle,erlotinib (25 mg/kg/day), bortezomib (0.25 mg/kg), the combination oferlotinib and bortezomib. Tumor dimensions are reported as the foldchange relative to size of the same tumor on Day 1. *P=x using a one wayANOVA test. n=8 mice per group. (e) Confocal microscopy images ofcleaved caspase 3 (red) and DNA (TOPRO-3, blue) in tumor biopsies fromxenografts tumors used in (d) treated with vehicle, erlotinib,bortezomib or bortezomib and erlotinib in combo. Scale bar, 20 μm.

REFERENCES Example 3

-   1. Desgrosellier, J. S. & Cheresh, D. A. Integrins in cancer:    biological implications and therapeutic opportunities. Nat Rev    Cancer 10, 9-22 (2010).-   2. Singh, A. & Settleman, J. EMT, cancer stem cells and drug    resistance: an emerging axis of evil in the war on cancer. Oncogene    29, 4741-4751 (2010).-   3. Lo, P. K., et al. CD49f and CD61 identify Her2/neu-induced    mammary tumor-initiating cells that are potentially derived from    luminal progenitors and maintained by the integrin-TGFbeta    signaling. Oncogene (2011).-   4. Vaillant, F., et al. The mammary progenitor marker CD61/beta3    integrin identifies cancer stem cells in mouse models of mammary    tumorigenesis. Cancer Res 68, 7711-7717 (2008).-   5. Galliher, A. J. & Schiemann, W. P. Beta3 integrin and Src    facilitate transforming growth factor-beta mediated induction of    epithelial-mesenchymal transition in mammary epithelial cells.    Breast cancer research: BCR 8, R42 (2006).-   6. Mamuya, F. A. & Duncan, M. K. aV integrins and TGF-beta-induced    EMT: a circle of regulation. Journal of cellular and molecular    medicine 16, 445-455 (2012).-   7. Desgrosellier, J. S., et al. An integrin alpha(v)beta(3)-c-Src    oncogenic unit promotes anchorage-independence and tumor    progression. Nat Med 15, 1163-1169 (2009).-   8. Boudreau, N., et al. Induction of the angiogenic phenotype by Hox    D3. J Cell Biol 139, 257-264 (1997).-   9. Dean, M., Fojo, T. & Bates, S. Tumour stem cells and drug    resistance. Nature Reviews Cancer 5, 275-284 (2005).-   10. Martin, K. H., et al. Integrin Connections Map: To Infinity and    Beyond. Science 296, 1652-1653 (2002).-   11. Newlaczyl, A. U. & Yu, L. G. Galectin-3—a jack-of-all-trades in    cancer. Cancer letters 313, 123-128 (2011).-   12. Shalom-Feuerstein, R., et al. K-ras nanoclustering is subverted    by overexpression of the scaffold protein galectin-3. Cancer    research 68, 6608-6616 (2008).-   13. Markowska, A. I., Liu, F. T. & Panjwani, N. Galectin-3 is an    important mediator of VEGF- and bFGF-mediated angiogenic response. J    Exp Med 207, 1981-1993 (2010).-   14. Pylayeva-Gupta, Y., Grabocka, E. & Bar-Sagi, D. RAS oncogenes:    weaving a tumorigenic web. Nat Rev Cancer 11, 761-774 (2011).-   15. Delhase, M., et al. TANK-binding kinase 1 (TBK1) controls cell    survival through PAI-2/serpinB2 and transglutaminase 2. Proceedings    of the National Academy of Sciences of the United States of America    109, E177-186 (2012).-   16. Jinushi, M., et al. ATM-mediated DNA damage signals mediate    immune escape through integrin-alphavbeta3-dependent mechanisms.    Cancer Res 72, 56-65 (2012).-   17. Schmeichel, K. L. & Bissell, M. J. Modeling tissue-specific    signaling and organ function in three dimensions. Journal of cell    science 116, 2377-2388 (2003).-   18. Singh, A., et al. A gene expression signature associated with    “K-Ras addiction” reveals regulators of EMT and tumor cell survival.    Cancer Cell 15, 489-500 (2009).

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A method for: overcoming or diminishing or preventing a Growth FactorInhibitor (GFI) resistance in a cell, or increasing thegrowth-inhibiting effectiveness of a Growth Factor inhibitor on a cell,or sensitizing, increasing sensitivity to or re-sensitizing a cell to aGrowth Factor Inhibitor (GFI), or sensitizing, increasing sensitivity toor re-sensitizing a dysfunctional cell, a tumor or cancer to a drug,sensitizing, increasing sensitivity to or re-sensitizing a tumor that isresistant to a cancer or anti-tumor drug, or reversing a tumor cell, acancer cell, a cancer stem cell or a dysfunctional cell initiation orself-renewal capacity, wherein optionally the cell is a tumor cell, acancer cell, a cancer stem cell, or a dysfunctional cell, the methodcomprising: (a) (1) providing at least one compound, composition orformulation comprising: (i) an inhibitor or depleter of integrin α_(v)β₃(anb3), or an inhibitor of integrin α_(v)β₃ (anb3) protein activity, oran inhibitor of the formation or activity of an integrin anb3/RalBsignaling complex, or an inhibitor of the formation or signalingactivity of an integrin α_(v)β₃ (anb3)/RalB/NFkB signaling axis; (ii) aninhibitor or depleter of a RalB protein or an inhibitor of a RalBprotein activation, or an inhibitor or depleter of the recruitment ofKRAS/RalB to the plasma membrane or the association of KRAS to RalB;(iii) an inhibitor or depleter of a Src or a Tank Binding Kinase (TBK1)protein or an inhibitor of Src or TBK1 protein activation, whereinoptionally the inhibitor of the Src or the TBK1 protein activity is: anamlexanox (or2-amino-7-isopropyl-5-oxo-5H-chromeno[2,3-b]pyridine-3-carboxylic acid),or an APHTHASOL™; or a γ(1)34.5 protein of herpes simplex viruses (HSV);or, BX795; or an azabenzimidazole or an analog or derivative thereof; ora 6-amino-pyrazolopyrimidine or an analog or derivative thereof; or, acompound having one of the following formulas, or an analog orderivative thereof: Molecule IKKε TBK1 IKKβ IKKα

0.77 0.44 >10 >10

>10 0.50 >10 >10

>10 0.64 8.76 >10

>10 0.67 >10 >10

>10 0.87 >10 >10

and optionally the inhibitor of the Src or the TBK1 protein activity isan allosteric inhibitor of Src or TBK1 protein activity; (iv) aninhibitor or depleter of a NFKB or a Interferon regulatory factor 3(IRF3) protein or an inhibitor of RalB protein activation, whereinoptionally the inhibitor of the NFKB or the IRF3 protein activity is anallosteric inhibitor of an NFKB or an Interferon regulatory factor 3(IRF3) protein activity; (v) an inhibitor or depleter of NFKB or IKK, oran inhibitor of NFKB or IKK protein activation, wherein optionally theNFKB inhibitor comprises a lenalidomide or a REVLIMID™ (Celgene Corp.,Summit, N.J.) and optionally the IKK inhibitor comprises a PS1145(Millennium Pharmaceuticals, Cambridge, Mass.); (vi) a lenalidomide or aREVLIMID™ and PS1145; (vii) a lenalidomide or a REVLIMID™; a PS1145;and, a Receptor Tyrosine Kinase (RTK) inhibitor, and optionally the RTKinhibitor comprises SU14813 (Pfizer, San Diego, Calif.); (viii) aninhibitor of Galectin-3; or (ix) any combination of (i) to (viii), or(2) one or any combination of the compound, composition or formulation,or compounds, compositions or formulations, of (1), and at least onegrowth factor inhibitor; and (b) administering a sufficient amount ofthe at least one compound, composition or formulation to the cell, orthe combination of compounds, to: overcome or diminish or prevent aGrowth Factor Inhibitor (GFI) resistance in a cell, or increase thegrowth-inhibiting effectiveness of a Growth Factor inhibitor on a cell,or sensitize, increase sensitivity or re-sensitize a cell to a GrowthFactor Inhibitor (GFI), or sensitize, increase sensitivity orre-sensitize a dysfunctional cell, a tumor or cancer to a drug, whereinoptionally the drug is a Receptor Tyrosine Kinase (RTK) inhibitor, or anerlotinib, a lapatinib or a lenalidomide, sensitize, increasesensitivity or re-sensitize a tumor that is resistant to a cancer oranti-tumor drug, or reverse a tumor cell, a cancer cell, a cancer stemcell or a dysfunctional cell initiation or self-renewal capacity.
 2. Themethod of claim 1, wherein: (a) the at least one compound, compositionor formulation, or combination of compounds, is formulated as apharmaceutical composition; (b) the method of (a), wherein the compound,composition or formulation or pharmaceutical composition is administeredin vitro, ex vivo or in vivo, or is administered to an individual inneed thereof; or (c) the method of (a) or (b), wherein the at least onecompound, composition or formulation is a pharmaceutical composition isformulated for administration intravenously (IV), parenterally, nasally,topically, orally, or by liposome or targeted or vessel-targetednanoparticle delivery.
 3. A kit, a blister package, a lidded blister ora blister card or packet, a clamshell, a tray or a shrink wrap,comprising; (a) at least one compound, composition or formulation usedto practice the method of claim 1, and (b) at least one Growth FactorInhibitor, wherein optionally the Growth Factor Inhibitor is orcomprises an anti-metabolite inhibitor, a gemcitabine, GEMZAR™, amitotic poison, a paclitaxel, a taxol, ABRAXANE™, an erlotinib,TARCEVA™, a lapatinib, TYKERB™, or an insulin growth factor inhibitor,or any combination thereof; or, the Growth Factor Inhibitor decreases,slows or blocks new blood vessel growth, neovascularization orangiogenesis; or, wherein administering the Growth Factor Inhibitortreats or ameliorates conditions that are responsive to blocking orslowing cell growth, and/or the development of neovascularization or newblood vessels.
 4. A method for determining: whether an individual or apatient would benefit from or respond to administration of a GrowthFactor Inhibitor, or which individuals or patients would benefit from acombinatorial approach comprising administration of a combination of: atleast one growth factor and at least one compound, composition orformulation used to practice the method of claim 1, such as an NfKbinhibitor, the method comprising: detecting the levels or amount ofintegrin α_(v)β₃ (anb3) and/or active RalB complex in or on a cell, atissue or a tissue sample, wherein optionally the detection is byanalysis or visualization of a biopsy or a tissue, urine, fluid, serumor blood sample, or a pathology slide taken from the patient orindividual, or by a fluorescence-activated cell sorting (FACS) or flowcytometry analysis or the sample or biopsy, wherein optionally the cellor tissue or tissue sample is or is derived from a tumor or a cancer,wherein optionally the method further comprises taking a biopsy or atissue, urine, fluid, serum or blood sample from an individual or apatient, wherein a finding of increased levels or amounts of integrinα_(v)β₃ (anb3) and/or active RalB complexes in or on the cell, tissue orthe tissue sample as compared to normal, normalized or wild type cellsor tissues, indicates that: the individual or patient would benefit froma combinatorial approach comprising administration of a combination of:at least one growth factor and at least one compound, composition orformulation used to practice the method of claim
 1. 5. The method ofclaim 4, wherein the detecting of the levels or amount of integrinα_(v)β₃ (anb3) and/or active RalB complex in or on the cell, tissue orthe tissue sample is done before or during a drug or a pharmaceuticaltreatment of an individual using at least one compound, composition orformulation used to practice the method of claim
 1. 6. (canceled)
 7. Atherapeutic combination of drugs comprising a combination of at leasttwo compounds: wherein the at least two compounds comprise or consistof: (1) at least one compound comprising: (i) an inhibitor or depleterof integrin α_(v)β₃ (anb3), or an inhibitor of integrin α_(v)β₃ (anb3)protein activity, or an inhibitor of the formation or activity of anintegrin anb3/RalB signaling complex, or an inhibitor of the formationor signaling activity of an integrin α_(v)β₃ (anb3)/RalB/NFkB signalingaxis, wherein optionally the inhibitor of integrin α_(v)β₃ proteinactivity is an allosteric inhibitor of integrin α_(v)β₃ proteinactivity; (ii) an inhibitor or depleter of a RalB protein or aninhibitor of a RalB protein activation, or an inhibitor or depleter ofthe recruitment of KRAS/RalB to the plasma membrane or the associationof KRAS to RalB, wherein optionally the inhibitor is an allostericinhibitor, or the inhibitor of the RalB protein activity is anallosteric inhibitor of RalB protein activity; (iii) an inhibitor ordepleter of a Src or a Tank Binding Kinase (TBK1) protein or aninhibitor of Src or TBK1 protein activation, wherein optionally theinhibitor of the Src or the TBK1 protein activity is: an amlexanox (or2-amino-7-isopropyl-5-oxo-5H-chromeno[2,3-b]pyridine-3-carboxylic acid),or an APHTHASOL™; or a γ(1)34.5 protein of herpes simplex viruses (HSV);or, BX795; or an azabenzimidazole or an analog or derivative thereof; ora 6-amino-pyrazolopyrimidine or an analog or derivative thereof; or, acompound having one of the following formulas, or an analog orderivative thereof: Molecule IKKε TBK1 IKKβ IKKα

0.77 0.44 >10 >10

>10 0.50 >10 >10

>10 0.64 8.76 >10

>10 0.67 >10 >10

>10 0.87 >10 >10

and optionally the inhibitor of the Src or the TBK1 protein activity isan allosteric inhibitor of Src or TBK1 protein activity; (iv) aninhibitor or depleter of a NFKB or a Interferon regulatory factor 3(IRF3) protein or an inhibitor of RalB protein activation, whereinoptionally the inhibitor of the NFKB or the IRF3 protein activity is anallosteric inhibitor of an NFKB or an Interferon regulatory factor 3(IRF3) protein activity; (v) an inhibitor or depleter of NFKB or IKK, oran inhibitor of NFKB or IKK protein activation, wherein optionally theNFKB inhibitor comprises a lenalidomide or a REVLIMID™ (Celgene Corp.,Summit, N.J.) and optionally the IKK inhibitor comprises a PS1145(Millennium Pharmaceuticals, Cambridge, Mass.); (vi) a lenalidomide or aREVLIMID™ and PS1145; (vii) a lenalidomide or a REVLIMID™; a PS1145;and, a Receptor Tyrosine Kinase (RTK) inhibitor, and optionally the RTKinhibitor comprises SU14813 (Pfizer, San Diego, Calif.); (viii) aninhibitor of Galectin-3; or (ix) any combination of (i) to (viii), or(2) one or any combination of the compound, composition or formulation,or compounds, compositions or formulations, of (1), and at least onegrowth factor inhibitor, wherein optionally the at least one growthfactor inhibitor comprises a Receptor Tyrosine Kinase (RTK) inhibitor, aSrc inhibitor, an anti-metabolite inhibitor, a gemcitabine, a GEMZAR™, amitotic poison, a paclitaxel, a taxol, an ABRAXANE™, an erlotinib, aTARCEVA™, a lapatinib, a TYKERB™, a cetuxamib, an ERBITUX™, or aninsulin growth factor inhibitor; wherein optionally the combination orthe therapeutic combination comprises: (i) an inhibitor or depleter of aSrc or a Tank Binding Kinase-1 (TBK1) protein or an inhibitor of Src orTBK1 protein activation, wherein optionally the inhibitor of the Src orthe TBK1 protein activity is an amlexanox (or2-amino-7-isopropyl-5-oxo-5H-chromeno[2,3-b]pyridine-3-carboxylic acid)or APHTHASOL™, and (ii) an RTK inhibitor, wherein optionally the RTKinhibitor is a Src inhibitor, an anti-metabolite inhibitor, agemcitabine, a GEMZAR™, a mitotic poison, a paclitaxel, a taxol, anABRAXANE™, an erlotinib, a TARCEVA™, a lapatinib, a TYKERB™, acetuxamib, an ERBITUX™, or an insulin growth factor inhibitor or acombination thereof; wherein optionally the combination or thetherapeutic combination comprises an erlotinib with either aLenalidomide or a PS-1145, or both a Lenalidomide and a PS-1145.
 8. Acombination, or a therapeutic combination, for overcoming or diminishingor preventing Growth Factor Inhibitor (GFI) resistance in a cell, or, amethod for increasing the growth-inhibiting effectiveness of a GrowthFactor inhibitor on a cell, or, a method for re-sensitizing a cell to aGrowth Factor Inhibitor (GFI), wherein the combination comprises orconsists of: (1) at least one compound comprising or consisting of: (i)an inhibitor or depleter of integrin α_(v)β₃ (anb3), or an inhibitor ofintegrin α_(v)β₃ (anb3) protein activity, or an inhibitor of theformation or activity of an integrin anb3/RalB signaling complex, or aninhibitor of the formation or signaling activity of an integrin α_(v)β₃(anb3)/RalB/NFkB signaling axis, wherein optionally the inhibitor ofintegrin α_(v)β₃ protein activity is an allosteric inhibitor of integrinα_(v)β₃ protein activity; (ii) an inhibitor or depleter of a RalBprotein or an inhibitor of a RalB protein activation, or an inhibitor ordepleter of the recruitment of KRAS/RalB to the plasma membrane or theassociation of KRAS to RalB, wherein optionally the inhibitor is anallosteric inhibitor, or the inhibitor of the RalB protein activity isan allosteric inhibitor of RalB protein activity; (iii) an inhibitor ordepleter of a Src or a Tank Binding Kinase (TBK1) protein or aninhibitor of Src or TBK1 protein activation, wherein optionally theinhibitor of the Src or the TBK1 protein activity is: an amlexanox (or2-amino-7-isopropyl-5-oxo-5H-chromeno[2,3-b]pyridine-3-carboxylic acid),or an APHTHASOL™; or a γ(1)34.5 protein of herpes simplex viruses (HSV);or, BX795; or an azabenzimidazole or an analog or derivative thereof; ora 6-amino-pyrazolopyrimidine or an analog or derivative thereof; or, acompound having one of the following formulas, or an analog orderivative thereof: Molecule IKKε TBK1 IKKβ IKKα

0.77 0.44 >10 >10

>10 0.50 >10 >10

>10 0.64 8.76 >10

>10 0.67 >10 >10

>10 0.87 >10 >10

and optionally the inhibitor of the Src or the TBK1 protein activity isan allosteric inhibitor of Src or TBK1 protein activity; (iv) aninhibitor or depleter of a NFKB or a Interferon regulatory factor 3(IRF3) protein or an inhibitor of RalB protein activation, whereinoptionally the inhibitor of the NFKB or the IRF3 protein activity is anallosteric inhibitor of an NFKB or an Interferon regulatory factor 3(IRF3) protein activity; (v) an inhibitor or depleter of NFKB or IKK, oran inhibitor of NFKB or IKK protein activation, wherein optionally theNFKB inhibitor comprises a lenalidomide or a REVLIMID™ (Celgene Corp.,Summit, N.J.) and optionally the IKK inhibitor comprises a PS1145(Millennium Pharmaceuticals, Cambridge, Mass.); (vi) a lenalidomide or aREVLIMID™ and PS1145; (vii) a lenalidomide or a REVLIMID™; a PS1145;and, a Receptor Tyrosine Kinase (RTK) inhibitor, and optionally the RTKinhibitor comprises SU14813 (Pfizer, San Diego, Calif.); (viii) aninhibitor of Galectin-3; or (ix) any combination of (i) to (viii), or(2) one or any combination of the compound, composition or formulation,or compounds, compositions or formulations, of (1), and at least onegrowth factor inhibitor, wherein optionally the at least one growthfactor inhibitor comprises a Receptor Tyrosine Kinase (RTK) inhibitor, aSrc inhibitor, an anti-metabolite inhibitor, a gemcitabine, a GEMZAR™, amitotic poison, a paclitaxel, a taxol, an ABRAXANE™, an erlotinib, aTARCEVA™, a lapatinib, a TYKERB™, a cetuxamib, an ERBITUX™, or aninsulin growth factor inhibitor; wherein optionally the combination orthe therapeutic combination comprises: (i) an inhibitor or depleter of aSrc or a Tank Binding Kinase-1 (TBK1) protein or an inhibitor of Src orTBK1 protein activation, wherein optionally the inhibitor of the Src orthe TBK1 protein activity is an amlexanox (or2-amino-7-isopropyl-5-oxo-5H-chromeno[2,3-b]pyridine-3-carboxylic acid)or APHTHASOL™, and (ii) an RTK inhibitor, wherein optionally the RTKinhibitor is a Src inhibitor, an anti-metabolite inhibitor, agemcitabine, a GEMZAR™, a mitotic poison, a paclitaxel, a taxol, anABRAXANE™, an erlotinib, a TARCEVA™, a lapatinib, a TYKERB™, acetuxamib, an ERBITUX™, or an insulin growth factor inhibitor or acombination thereof; wherein optionally the combination or thetherapeutic combination comprises an erlotinib with either aLenalidomide or a PS-1145, or both a Lenalidomide and a PS-1145.
 9. Themethod of claim 1, wherein the method comprises sensitizing, increasingsensitivity to or re-sensitizing a dysfunctional cell, a tumor or cancerto a drug comprising a Receptor Tyrosine Kinase (RTK) inhibitor, anEGFR1 inhibitor, an EGFR1/EGFR2 inhibitor or an IGF-1R inhibitor, or anerlotinib, a linsitinib, a lapatinib or a lenalidomide.
 10. The methodof claim 1, wherein the method comprises providing at least onecompound, composition or formulation comprising an inhibitor of integrinα_(v)β₃ protein activity that is an allosteric inhibitor of integrinα_(v)β₃ protein activity.
 11. The method of claim 1, wherein the methodcomprises providing at least one compound, composition or formulationcomprising an allosteric inhibitor or depleter of a RalB protein or anallosteric inhibitor of a RalB protein activation, or an allostericinhibitor or depleter of the recruitment of KRAS/RalB to the plasmamembrane or the association of KRAS to RalB.
 12. The method of claim 1,wherein the method comprises providing at least one growth factorinhibitor comprising a Receptor Tyrosine Kinase (RTK) inhibitor, a Srcinhibitor, an anti-metabolite inhibitor, a gemcitabine, a GEMZAR™, amitotic poison, a paclitaxel, a taxol, an ABRAXANE™, an erlotinib, aTARCEVA™, a lapatinib, a TYKERB™, a cetuxamib, an ERBITUX™, or aninsulin growth factor inhibitor.
 13. The method of claim 1, wherein themethod comprises providing a combination or the therapeutic combinationcomprising: (i) an inhibitor or depleter of a Src or a Tank BindingKinase-1 (TBK1) protein or an inhibitor of Src or TBK1 proteinactivation, wherein optionally the inhibitor of the Src or the TBK1protein activity is an amlexanox (or2-amino-7-isopropyl-5-oxo-5H-chromeno[2,3-b]pyridine-3-carboxylic acid)or APHTHASOL™, and (ii) an RTK inhibitor, wherein optionally the RTKinhibitor is a Src inhibitor, an anti-metabolite inhibitor, agemcitabine, a GEMZAR™, a mitotic poison, a paclitaxel, a taxol, anABRAXANE™ an erlotinib, a TARCEVA™, a lapatinib, a TYKERB™, a cetuxamib,an ERBITUX™, or an insulin growth factor inhibitor or a combinationthereof.
 14. The method of claim 1, wherein the method comprisesproviding a combination or the therapeutic combination comprising anerlotinib with either a Lenalidomide or a PS-1145, or both aLenalidomide and a PS-1145.
 15. The method of claim 1, wherein the atleast one compound, composition or formulation comprises or is aninhibitor of transcription, translation or protein expression.
 16. Themethod of claim 1, wherein the at least one compound, composition orformulation comprises is a small molecule, a protein, an antibody, amonoclonal antibody, a nucleic acid, a lipid or a fat, a polysaccharide,an RNA or a DNA.
 16. The method of claim 1, wherein the at least onecompound, composition or formulation comprises: a VITAXIN™ (AppliedMolecular Evolution, San Diego, Calif.) antibody, a humanized version ofan LM609 monoclonal antibody, an LM609 monoclonal antibody, or anyantibody that functionally blocks an α_(v)β₃ integrin or any member ofan α_(v)β₃ integrin-comprising complex or an integrin α_(v)β₃(anb3)/RalB/NFkB signaling axis.
 17. The method of claim 1, wherein theat least one compound, composition or formulation comprises or is a Srcinhibitor, a dasatinib, a saracatinib; a bosutinib; a NVP-BHG712, or anycombination thereof.
 18. The method of claim 1, wherein the GrowthFactor Inhibitor is or comprises an anti-metabolite inhibitor, agemcitabine, GEMZAR™, a mitotic poison, a paclitaxel, a taxol,ABRAXANE™, an erlotinib, TARCEVA™, a lapatinib, TYKERB™, or an insulingrowth factor inhibitor, or any combination thereof.
 19. The method ofclaim 1, wherein the Growth Factor Inhibitor decreases, slows or blocksnew blood vessel growth, neovascularization or angiogenesis; or, whereinadministering the Growth Factor Inhibitor treats or amelioratesconditions that are responsive to blocking or slowing cell growth,and/or the development of neovascularization or new blood vessels. 20.The method of claim 1, wherein (a) the NF-kB inhibitor comprises orconsists of one or more of: an antioxidant; an α-lipoic acid; anα-tocopherol; a 2-amino-1-methyl-6-phenylimidazo[4,5-β]pyridine; anallopurinol; an anetholdithiolthione; a cepharanthine; a beta-carotene;a dehydroepiandrosterone (DHEA) or a DHEA-sulfate (DHEAS); adimethyldithiocarbamates (DMDTC); a dimethylsulfoxide (DMSO); a flavone,a Glutathione; Vitamin C or Vitamin B6, or one or more compositionslisted in Table 1 or Table 2, or any combination thereof; (b) the atleast one compound, composition or formulation, or combination ofcompounds, comprises a proteasome inhibitor or a protease inhibitor thatcan inhibit an Rel and/or an NFkB, or one or more compositions listed inTable 2, or any combination thereof; or (c) the at least one compound,composition or formulation, or combination of compounds, comprises anIκBa (nuclear factor of kappa light polypeptide gene enhancer in B-cellsinhibitor, alpha) phosphorylation and/or degradation inhibitor, or oneor more compositions listed in Table 3, or any combination thereof. 21.The method of claim 1, wherein the method reduces, treats or amelioratesthe level of disease in a retinal age-related macular degeneration, adiabetic retinopathy, a cancer or carcinoma, a glioblastoma, a neuroma,a neuroblastoma, a colon carcinoma, a hemangioma, an infection and/or acondition with at least one inflammatory component, and/or anyinfectious or inflammatory disease, such as a rheumatoid arthritis, apsoriasis, a fibrosis, leprosy, multiple sclerosis, inflammatory boweldisease, or ulcerative colitis or Crohn's disease.